CA1323550C - Histological fixatives - Google Patents
Histological fixativesInfo
- Publication number
- CA1323550C CA1323550C CA000579772A CA579772A CA1323550C CA 1323550 C CA1323550 C CA 1323550C CA 000579772 A CA000579772 A CA 000579772A CA 579772 A CA579772 A CA 579772A CA 1323550 C CA1323550 C CA 1323550C
- Authority
- CA
- Canada
- Prior art keywords
- fixative
- group
- member selected
- chloride
- per liter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000000834 fixative Substances 0.000 title claims abstract description 83
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 34
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 33
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000002253 acid Substances 0.000 claims abstract description 14
- 150000003839 salts Chemical class 0.000 claims abstract description 14
- 150000002009 diols Chemical class 0.000 claims abstract description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 12
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 claims abstract description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910052753 mercury Inorganic materials 0.000 claims abstract description 11
- 150000004072 triols Chemical class 0.000 claims abstract description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 235000019253 formic acid Nutrition 0.000 claims abstract description 6
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 6
- 230000003647 oxidation Effects 0.000 claims abstract description 6
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 6
- 239000011780 sodium chloride Substances 0.000 claims abstract description 6
- 150000007513 acids Chemical class 0.000 claims abstract description 5
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 34
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- 238000010186 staining Methods 0.000 claims description 18
- 239000011592 zinc chloride Substances 0.000 claims description 17
- 235000005074 zinc chloride Nutrition 0.000 claims description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 9
- 239000002357 osmotic agent Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 6
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 5
- SOCTUWSJJQCPFX-UHFFFAOYSA-N dichromate(2-) Chemical compound [O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O SOCTUWSJJQCPFX-UHFFFAOYSA-N 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 230000035515 penetration Effects 0.000 claims description 3
- 238000000034 method Methods 0.000 claims 11
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims 4
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 claims 4
- 229910001626 barium chloride Inorganic materials 0.000 claims 4
- 239000001110 calcium chloride Substances 0.000 claims 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims 4
- 229960000359 chromic chloride Drugs 0.000 claims 4
- LJAOOBNHPFKCDR-UHFFFAOYSA-K chromium(3+) trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Cl-].[Cr+3] LJAOOBNHPFKCDR-UHFFFAOYSA-K 0.000 claims 4
- 239000011636 chromium(III) chloride Substances 0.000 claims 4
- 235000007831 chromium(III) chloride Nutrition 0.000 claims 4
- 229910001631 strontium chloride Inorganic materials 0.000 claims 4
- AHBGXTDRMVNFER-UHFFFAOYSA-L strontium dichloride Chemical compound [Cl-].[Cl-].[Sr+2] AHBGXTDRMVNFER-UHFFFAOYSA-L 0.000 claims 4
- 229920002307 Dextran Polymers 0.000 claims 3
- 150000002016 disaccharides Chemical class 0.000 claims 3
- 150000002772 monosaccharides Chemical class 0.000 claims 3
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims 3
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims 2
- 230000000877 morphologic effect Effects 0.000 claims 2
- 102000004169 proteins and genes Human genes 0.000 claims 2
- 108090000623 proteins and genes Proteins 0.000 claims 2
- 229920003169 water-soluble polymer Polymers 0.000 claims 2
- 239000012024 dehydrating agents Substances 0.000 claims 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 1
- 229920000642 polymer Polymers 0.000 claims 1
- 125000000185 sucrose group Chemical group 0.000 claims 1
- 239000007864 aqueous solution Substances 0.000 abstract description 4
- 239000013543 active substance Substances 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- -1 ethylene glycol Chemical class 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 44
- 239000000243 solution Substances 0.000 description 6
- 238000009472 formulation Methods 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 4
- 238000007654 immersion Methods 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 101710105077 Agglutinin-1 Proteins 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 240000003864 Ulex europaeus Species 0.000 description 2
- 235000010730 Ulex europaeus Nutrition 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000001744 histochemical effect Effects 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 229960002523 mercuric chloride Drugs 0.000 description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000002741 palatine tonsil Anatomy 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- OFNXOACBUMGOPC-HZYVHMACSA-N 5'-hydroxystreptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](CO)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O OFNXOACBUMGOPC-HZYVHMACSA-N 0.000 description 1
- 241000269350 Anura Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241000078511 Microtome Species 0.000 description 1
- 108010052989 Naphthol AS D Esterase Proteins 0.000 description 1
- 108010081750 Reticulin Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229940075397 calomel Drugs 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical compound Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 229960001484 edetic acid Drugs 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- OFNXOACBUMGOPC-UHFFFAOYSA-N hydroxystreptomycin Natural products CNC1C(O)C(O)C(CO)OC1OC1C(C=O)(O)C(CO)OC1OC1C(N=C(N)N)C(O)C(N=C(N)N)C(O)C1O OFNXOACBUMGOPC-UHFFFAOYSA-N 0.000 description 1
- OKPOKMCPHKVCPP-UHFFFAOYSA-N isoorientaline Natural products C1=C(O)C(OC)=CC(CC2C3=CC(OC)=C(O)C=C3CCN2C)=C1 OKPOKMCPHKVCPP-UHFFFAOYSA-N 0.000 description 1
- 239000003041 laboratory chemical Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 229940008718 metallic mercury Drugs 0.000 description 1
- DWCZIOOZPIDHAB-UHFFFAOYSA-L methyl green Chemical compound [Cl-].[Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)[N+](C)(C)C)=C1C=CC(=[N+](C)C)C=C1 DWCZIOOZPIDHAB-UHFFFAOYSA-L 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- INCIMLINXXICKS-UHFFFAOYSA-M pyronin Y Chemical compound [Cl-].C1=CC(=[N+](C)C)C=C2OC3=CC(N(C)C)=CC=C3C=C21 INCIMLINXXICKS-UHFFFAOYSA-M 0.000 description 1
- JTQHYPFKHZLTSH-UHFFFAOYSA-N reticulin Natural products COC1CC(OC2C(CO)OC(OC3C(O)CC(OC4C(C)OC(CC4OC)OC5CCC6(C)C7CCC8(C)C(CCC8(O)C7CC=C6C5)C(C)O)OC3C)C(O)C2OC)OC(C)C1O JTQHYPFKHZLTSH-UHFFFAOYSA-N 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 229910052712 strontium Inorganic materials 0.000 description 1
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
- G01N2474/20—Immunohistochemistry assay
Abstract
HISTOLOGICAL FIXATIVE
ABSTRACT OF THE INVENTION
A mercury and formaldehyde free histological fixative.
The fixative employs standard non-toxic components comprising one or more alkanols, one or more diols and triols such as ethylene glycol, and one or more acids such as acetic and formic acid in an aqueous solution. A salt of a metal ion having an oxidation state of at least two may be added as an optional mordant. Osmotically active substances such as sodium chloride may be used as an option as desired to control osmotically induced cell volume changes.
ABSTRACT OF THE INVENTION
A mercury and formaldehyde free histological fixative.
The fixative employs standard non-toxic components comprising one or more alkanols, one or more diols and triols such as ethylene glycol, and one or more acids such as acetic and formic acid in an aqueous solution. A salt of a metal ion having an oxidation state of at least two may be added as an optional mordant. Osmotically active substances such as sodium chloride may be used as an option as desired to control osmotically induced cell volume changes.
Description
BACKGROUND OF Tll~_INVENTION 13 2 3 ~ ~ O
In the past, various formulations have been employed as histological fixatives to provide microscopic detail to sec-tions of biological tissue. One such commonly used fixative is that known as B-S, which is an aqueous solution of mercuric chloride, sodium acetate and formaldehyde. Disadvantages of this fixative include dangerous exposure of laboratory person-nel to toxic mercury and formaldehyde and the unpleasant odor of the latter, expcnse of disposing of the fixative in a safe manner because of its mercury content, excessive wear on micro-tome blades due to the hardening of the tissue by mercury and the necessity of removing elememtal mercury from tissue sec-tions by treatment wi.th iodine. Due ~o precipitation of calomel and metallic mercury upon standing, fresh solutions must be prepared daily and any excess solutions create disposal prob-lS lems.
The various disadvantages associated with the presence of mercury preclude the use of n-5 fixative as a routine fixative and allow its use on]y in situations where microscopic detail is of the greatest sigl-ificance and importance, for example in the classification of a ma].ignancy diagnosed as a malignant lymphoma. Fortunately, the handling of tissue foll~owing fixa-tion by ~-5 is almost the same as that of tissue fixed in a routine fixative, such as neutral buffered formalin, except for the use of iodine.
While various other fixatives for general use have been proposed that are mercury and formaldehyde free~these have generally re~uired the use of special non-standard chemicals and present staining problems of one type or another that do not lend them to widespread use. 13 2 3 ~ ~ ~
SUMMARY OF THE_ INVENTION
By means of this invention, there has been provided a histological fixative that is free of mercury and formaldehyde and is comprised of conventional components that are substantially non-toxic and relatively inexpensive. The term ~histological fixative~ as used hereln is employed to include biological preservative for anlmal and plant tissue, preserving or flxing biological specimens such as frogs or the llke for laboratory study and slmllar applications and other forms of preserving or fixing such tissue to include embalming. While for explanatory purpnse~ the use 1s described in connection with preserving or fixing tissue for microscopic study it will be understood the employment is not limited thereto. The fixative is produced as a clear solutlon with a pleasant odor and extended shelf life. The tissue fixed by the fixatlve may be handled ln the,same manner,as that treated by a routine fixative and yields mlcroscopic detail greater than or equal to that produced by B-5 fixative containing mercury and formaldehyde.
.
Ventilation considered appropriate for the usual histology laboratory is adequate to keep exposure to the fixative within safe limits. The non-aqueous components of this fixative were present in sufficlently low concentration and the pH is sufficiently high (greater than 2) to allow safe disposal 25 of small volumes of the fixative like those used under typical laboratory conditions (for example 500 ml. per day) in conventional~sewage systems. ~ satisfactory microscopic appearance is obtained in sections of tissue immersed in the fixative for up to eight weeks, far longer than the few hours 30 permitted by B-5 fixative.
The fixative of this invention constitutes a solution used as a histological fixative prior to appllcation o~ hematoxy-lin and eosin stains, special histochemical stains or immunohist-ochemical stains. The fixative comprlses a solvent of one or 35 more alkanols such as methanol, one or more dlols or triols such as ethylene glycol or glycerol, one or more acids such as -132~550 formic acid and/or acetic acid and other carboxylic acids, picric acid, perchloric acid and the like, and water. The sol-ute consists of mordants to enhance staining qualities and/or osmotically active agents to control osmotically induced cell volume changes and to preserve red blood cells. Examples of mordants and oqmotically active a~ents are zinc chloride and sodium chloride respectively. Zinc chloride may also be employed for the dual function of mordant and osmotically active s ~bg tance .
The fixative formulation i9 simply prepared from standard inexpensive laboratory chemicals and has a substantial shelf life. Handling and exposure represent no problems that are not encountered in a conventional laboratory. The fixative is em-ployed for a variety of tissues and application of different stains with a wide range of tissues can be effected with en-hanced specific staining and reduced non-specific staining.
DESCRIPTION OF THE INVENTION
The histological fixative of this invention is comprised of so1vent components of one or more alkanols such as methanol or ethanol in a total concentration of about 200 to 800 milli-liters per liter of solvent.
A further solvent component comprises one or more.diols and triols in a total concentration of about 150 to about 700 milliliters per liter of solvent. Exemplary of such diols and 2S triols are ethylene glycol, glycerol, propylene glycol and trimethylene glycol.
~323~0 In addition, one or more acids such as formic acid and acetic acid are employed at a total concentration of about o.ol to about 0.5 mole per liter of solvent. Water is added and constitutes the remainder of the solvent.
The solute consists of mordants, such as a salt with a metal ion having an oxidation state of two or more in a concen-tration of zero up to about 0.2 mole per liter of solvent.
Zinc chloride is preferred but other salts, including those of strontium, calcium, barium and chromiu~ may also be employed.
Also there may be employed picric acid and an alkali dichromate such as potassium dichromate.
In some instances of histological fixation of tissues, osmotically induced cell volume changes of the tissue may be encountered. In such cases, sodium chloride is included in the ormulation as an osmotically active substance. In addition, sugars snch as the polysaccharides, sucrose, glucose and the like may be employed for this purpose where desired.
Alkanoll diol and triol, acid and mordant components have been individually employed with other components in other for-mulations in preparing tissue for microscopy but have not been employed in applicant's novel formulation in the combination herein described. The individual components have met with ac-ceptance for their individual characteristics. Thus the alka-nols, particularly methanol and ethanol, are widely recognized as tissue fixatives. Generally, these alkanols are high concen-trations in an aqueous solution such as 70~ and 95~ for ethanol.
The diols and triols facilitate dehydration of the tissue and contribute to microscopic detail. Acetic acid is another acknow-1323~0 ledged fixative and also counteracts the tissue shrinkaqe pro-duced by the alkanols. Formic acid has been used primarily in decalcifying fixatives and has been found in this invention to provide satisfactory detail when used instead of acetic acid in the fixative. I~ater is necessary for optimal tissue preser-vation and increases the solubility of the zinc chloride or other salts in the fixative. Zinc chloride, or other appropriate salts as described above, is employed as a mordant.
~s a typical example of the histological fixative of this inventionJ there is listed below an exemplary formulation:
EXAMPLE
Ethanol, 400 mL/liter of fixative Ethylene glycol, 300 mL/liter of fixative Acetic acid, 10 mL/liter of fixative (0.17 mole per liter) Zinc chloride, 5.8 g/liter of fixative Water, remainder of fixative, i.e. about 290 mL/liter of fixative Various human tissues, including tonsil, vertebral bone marrow, and colon, were routinely immersed in the fixative for 1-8 hours, although immersion for up to 8 weeks gave a satis-factory microscopic appearance. Changes in tissue color and elasticity indicated rapid penetration, estimated as 2 mm/hour for tonsil. Gross tissue shrinkage was minimal. ~fter fixation, bone marrow was decalcified with an ethylene diamine tetraacetic acid solution or formic acid. It was found that postfixation of soft tissues for 2 hours in neutral buffered formalin did not alter the microscopic appearance of tissue sections or staining characteristics. Ilowever, 2 hours of immersion in an aqueous phosphate buffer in lieu of postfixation gave poor results. Following fixation and optional postfixation, the tissue was dehydrated by a graded series of ethanol solutions.
13235~0 followed by xylene and then embedded in paraffin. Paraffin in-filtrated the tissue thoroughly, producing tissue bloc~s with good cutting characteristics, including blocks containing large amounts of blood, fat, and dense smooth muscle such as uterine cervix.
Sections of tissue treated with this fixative had a satis-factory microscopic appearance using all of the following com-monly used stains. In general, the tissue showed the good mic-roscopic detail characteristic of B-5 fixative and the staining properties of a mordant-containing fixative such as Zen~er's, an aqueous solution of mercuric chloride, potassium dichromate, acetic acid, and optionally sodium sulfate. Comments on the microscopic appearance are included as appropriate. It is gen-- erally observed that compared to formalin fixation, this fixa-tive gives less non-specific staining, which tends to obscure or cloud the detecting stain.
STAINS
Giesma--satisfactory appearance ~ematoxylin and eosin--nuclear detail comparable to B-5 fixed tissue methyl green pyronin--less non-specific staining than formalin-fixed tissue periodic acid Schiff--less non-specific staining than formalin-fixed ~issue reticulin--little or no shrinkage was observed Ulex europaeus agglutinin I (binds to fucose)--enhanced staining compared to formalin-fixed tissue von Leder--less non-specific staining than formalin-fixed tissue The von Leder stain for naphthol AS-D chloroacetate ester-ase, which cannot satisfactorily be performed on tissue fixed by B-5, gives good results using tissue fixed by the fixative described herein. The fixative also allows accepta~le appearances of other histochemical stains as noted above, including periodic 13235~0 acid Schiff, methyl ~reen pyronin and reticulin. Immunoperoxi-dase staining for fucose, using Ulex europaeus agglutinin I, is enhanced in tissue fixed by this fixative as compared to tissue fixed by neutral buffered formalin.
Sections of tissue treated with this fixative for up to 2 weeks also had a satisfactory microscopic appearance using the following immunohistochemical stains. Again, comments on the microscopic appearance are included as appropriate.
collagen, type IV--satisfactory appearance common leukocyte antigen--enhanced staining compared to formalin-fixed tissue desmin-- staining is comparable to that of ethanol-fixed tissue (stain cannot be performed on formalin-fixed tissue) human epithelial keratin (AEl/AE3)--satisfactory appearance S-100-- no non-specific staining seen, unlike formalin-fixed tissue vimentin--less non-specific staining tha~ formalin-fixed tissue lS As an example of the use of this fixative, following immersion of tissue in it for at least one hour, conventional processing may be carried out in a closed machine or otherwise.
As an example, the tissue may be immersed in neutral buffered formalin, dehydrated ~y a series of aqueous ethanol solutions of gradually increasing concentration followed by xylene, in-filtrated by molten paraffin, embedded in paraffin, and finally sectioned. The section is placed on a glass slide, stained with conventional stains as exemplified above, and examined.micro-scopically. After examination, the slide may be preserved as in conventional practice.
Various changes and modifications may be made within this invention as will be apparent to those skilled in the art. Such changes and modifications are within the scope and teaching of this invention as defined in the claims appended hereto.
In the past, various formulations have been employed as histological fixatives to provide microscopic detail to sec-tions of biological tissue. One such commonly used fixative is that known as B-S, which is an aqueous solution of mercuric chloride, sodium acetate and formaldehyde. Disadvantages of this fixative include dangerous exposure of laboratory person-nel to toxic mercury and formaldehyde and the unpleasant odor of the latter, expcnse of disposing of the fixative in a safe manner because of its mercury content, excessive wear on micro-tome blades due to the hardening of the tissue by mercury and the necessity of removing elememtal mercury from tissue sec-tions by treatment wi.th iodine. Due ~o precipitation of calomel and metallic mercury upon standing, fresh solutions must be prepared daily and any excess solutions create disposal prob-lS lems.
The various disadvantages associated with the presence of mercury preclude the use of n-5 fixative as a routine fixative and allow its use on]y in situations where microscopic detail is of the greatest sigl-ificance and importance, for example in the classification of a ma].ignancy diagnosed as a malignant lymphoma. Fortunately, the handling of tissue foll~owing fixa-tion by ~-5 is almost the same as that of tissue fixed in a routine fixative, such as neutral buffered formalin, except for the use of iodine.
While various other fixatives for general use have been proposed that are mercury and formaldehyde free~these have generally re~uired the use of special non-standard chemicals and present staining problems of one type or another that do not lend them to widespread use. 13 2 3 ~ ~ ~
SUMMARY OF THE_ INVENTION
By means of this invention, there has been provided a histological fixative that is free of mercury and formaldehyde and is comprised of conventional components that are substantially non-toxic and relatively inexpensive. The term ~histological fixative~ as used hereln is employed to include biological preservative for anlmal and plant tissue, preserving or flxing biological specimens such as frogs or the llke for laboratory study and slmllar applications and other forms of preserving or fixing such tissue to include embalming. While for explanatory purpnse~ the use 1s described in connection with preserving or fixing tissue for microscopic study it will be understood the employment is not limited thereto. The fixative is produced as a clear solutlon with a pleasant odor and extended shelf life. The tissue fixed by the fixatlve may be handled ln the,same manner,as that treated by a routine fixative and yields mlcroscopic detail greater than or equal to that produced by B-5 fixative containing mercury and formaldehyde.
.
Ventilation considered appropriate for the usual histology laboratory is adequate to keep exposure to the fixative within safe limits. The non-aqueous components of this fixative were present in sufficlently low concentration and the pH is sufficiently high (greater than 2) to allow safe disposal 25 of small volumes of the fixative like those used under typical laboratory conditions (for example 500 ml. per day) in conventional~sewage systems. ~ satisfactory microscopic appearance is obtained in sections of tissue immersed in the fixative for up to eight weeks, far longer than the few hours 30 permitted by B-5 fixative.
The fixative of this invention constitutes a solution used as a histological fixative prior to appllcation o~ hematoxy-lin and eosin stains, special histochemical stains or immunohist-ochemical stains. The fixative comprlses a solvent of one or 35 more alkanols such as methanol, one or more dlols or triols such as ethylene glycol or glycerol, one or more acids such as -132~550 formic acid and/or acetic acid and other carboxylic acids, picric acid, perchloric acid and the like, and water. The sol-ute consists of mordants to enhance staining qualities and/or osmotically active agents to control osmotically induced cell volume changes and to preserve red blood cells. Examples of mordants and oqmotically active a~ents are zinc chloride and sodium chloride respectively. Zinc chloride may also be employed for the dual function of mordant and osmotically active s ~bg tance .
The fixative formulation i9 simply prepared from standard inexpensive laboratory chemicals and has a substantial shelf life. Handling and exposure represent no problems that are not encountered in a conventional laboratory. The fixative is em-ployed for a variety of tissues and application of different stains with a wide range of tissues can be effected with en-hanced specific staining and reduced non-specific staining.
DESCRIPTION OF THE INVENTION
The histological fixative of this invention is comprised of so1vent components of one or more alkanols such as methanol or ethanol in a total concentration of about 200 to 800 milli-liters per liter of solvent.
A further solvent component comprises one or more.diols and triols in a total concentration of about 150 to about 700 milliliters per liter of solvent. Exemplary of such diols and 2S triols are ethylene glycol, glycerol, propylene glycol and trimethylene glycol.
~323~0 In addition, one or more acids such as formic acid and acetic acid are employed at a total concentration of about o.ol to about 0.5 mole per liter of solvent. Water is added and constitutes the remainder of the solvent.
The solute consists of mordants, such as a salt with a metal ion having an oxidation state of two or more in a concen-tration of zero up to about 0.2 mole per liter of solvent.
Zinc chloride is preferred but other salts, including those of strontium, calcium, barium and chromiu~ may also be employed.
Also there may be employed picric acid and an alkali dichromate such as potassium dichromate.
In some instances of histological fixation of tissues, osmotically induced cell volume changes of the tissue may be encountered. In such cases, sodium chloride is included in the ormulation as an osmotically active substance. In addition, sugars snch as the polysaccharides, sucrose, glucose and the like may be employed for this purpose where desired.
Alkanoll diol and triol, acid and mordant components have been individually employed with other components in other for-mulations in preparing tissue for microscopy but have not been employed in applicant's novel formulation in the combination herein described. The individual components have met with ac-ceptance for their individual characteristics. Thus the alka-nols, particularly methanol and ethanol, are widely recognized as tissue fixatives. Generally, these alkanols are high concen-trations in an aqueous solution such as 70~ and 95~ for ethanol.
The diols and triols facilitate dehydration of the tissue and contribute to microscopic detail. Acetic acid is another acknow-1323~0 ledged fixative and also counteracts the tissue shrinkaqe pro-duced by the alkanols. Formic acid has been used primarily in decalcifying fixatives and has been found in this invention to provide satisfactory detail when used instead of acetic acid in the fixative. I~ater is necessary for optimal tissue preser-vation and increases the solubility of the zinc chloride or other salts in the fixative. Zinc chloride, or other appropriate salts as described above, is employed as a mordant.
~s a typical example of the histological fixative of this inventionJ there is listed below an exemplary formulation:
EXAMPLE
Ethanol, 400 mL/liter of fixative Ethylene glycol, 300 mL/liter of fixative Acetic acid, 10 mL/liter of fixative (0.17 mole per liter) Zinc chloride, 5.8 g/liter of fixative Water, remainder of fixative, i.e. about 290 mL/liter of fixative Various human tissues, including tonsil, vertebral bone marrow, and colon, were routinely immersed in the fixative for 1-8 hours, although immersion for up to 8 weeks gave a satis-factory microscopic appearance. Changes in tissue color and elasticity indicated rapid penetration, estimated as 2 mm/hour for tonsil. Gross tissue shrinkage was minimal. ~fter fixation, bone marrow was decalcified with an ethylene diamine tetraacetic acid solution or formic acid. It was found that postfixation of soft tissues for 2 hours in neutral buffered formalin did not alter the microscopic appearance of tissue sections or staining characteristics. Ilowever, 2 hours of immersion in an aqueous phosphate buffer in lieu of postfixation gave poor results. Following fixation and optional postfixation, the tissue was dehydrated by a graded series of ethanol solutions.
13235~0 followed by xylene and then embedded in paraffin. Paraffin in-filtrated the tissue thoroughly, producing tissue bloc~s with good cutting characteristics, including blocks containing large amounts of blood, fat, and dense smooth muscle such as uterine cervix.
Sections of tissue treated with this fixative had a satis-factory microscopic appearance using all of the following com-monly used stains. In general, the tissue showed the good mic-roscopic detail characteristic of B-5 fixative and the staining properties of a mordant-containing fixative such as Zen~er's, an aqueous solution of mercuric chloride, potassium dichromate, acetic acid, and optionally sodium sulfate. Comments on the microscopic appearance are included as appropriate. It is gen-- erally observed that compared to formalin fixation, this fixa-tive gives less non-specific staining, which tends to obscure or cloud the detecting stain.
STAINS
Giesma--satisfactory appearance ~ematoxylin and eosin--nuclear detail comparable to B-5 fixed tissue methyl green pyronin--less non-specific staining than formalin-fixed tissue periodic acid Schiff--less non-specific staining than formalin-fixed ~issue reticulin--little or no shrinkage was observed Ulex europaeus agglutinin I (binds to fucose)--enhanced staining compared to formalin-fixed tissue von Leder--less non-specific staining than formalin-fixed tissue The von Leder stain for naphthol AS-D chloroacetate ester-ase, which cannot satisfactorily be performed on tissue fixed by B-5, gives good results using tissue fixed by the fixative described herein. The fixative also allows accepta~le appearances of other histochemical stains as noted above, including periodic 13235~0 acid Schiff, methyl ~reen pyronin and reticulin. Immunoperoxi-dase staining for fucose, using Ulex europaeus agglutinin I, is enhanced in tissue fixed by this fixative as compared to tissue fixed by neutral buffered formalin.
Sections of tissue treated with this fixative for up to 2 weeks also had a satisfactory microscopic appearance using the following immunohistochemical stains. Again, comments on the microscopic appearance are included as appropriate.
collagen, type IV--satisfactory appearance common leukocyte antigen--enhanced staining compared to formalin-fixed tissue desmin-- staining is comparable to that of ethanol-fixed tissue (stain cannot be performed on formalin-fixed tissue) human epithelial keratin (AEl/AE3)--satisfactory appearance S-100-- no non-specific staining seen, unlike formalin-fixed tissue vimentin--less non-specific staining tha~ formalin-fixed tissue lS As an example of the use of this fixative, following immersion of tissue in it for at least one hour, conventional processing may be carried out in a closed machine or otherwise.
As an example, the tissue may be immersed in neutral buffered formalin, dehydrated ~y a series of aqueous ethanol solutions of gradually increasing concentration followed by xylene, in-filtrated by molten paraffin, embedded in paraffin, and finally sectioned. The section is placed on a glass slide, stained with conventional stains as exemplified above, and examined.micro-scopically. After examination, the slide may be preserved as in conventional practice.
Various changes and modifications may be made within this invention as will be apparent to those skilled in the art. Such changes and modifications are within the scope and teaching of this invention as defined in the claims appended hereto.
Claims (21)
1.
A mercury and formaldehyde free composition consisting essentially of an animal tissue histological fixative amount of a solution comprised of one or more alkanols as a tissue fixative, at least one member selected from the group consisting of an organic diol and triol having the capacity to dehydrate tissue and enhance microscopic detail, at least one acid to precipitate proteins, contribute to nuclear morphological detail, and increase penetration rate of fixative into tissue, 0 to about 700 millimoles per kilogram of solvent in the fixative of an osmotically active substance having the capacity to control osmotically induced cell volume changes, 0 to about 0.2 mole per liter of fixative of a mordant having the capacity to enhance staining characteristics, and water, the alkanols having a combined concentration of about 200 to about 800 milliliters per liter of fixative, the diols and triols having a combined concentration of about 150 to about 700 milliliters per liter of fixative, and the acids having a combined concentration of about 0.01 to about 0.5 mole per liter of fixative.
A mercury and formaldehyde free composition consisting essentially of an animal tissue histological fixative amount of a solution comprised of one or more alkanols as a tissue fixative, at least one member selected from the group consisting of an organic diol and triol having the capacity to dehydrate tissue and enhance microscopic detail, at least one acid to precipitate proteins, contribute to nuclear morphological detail, and increase penetration rate of fixative into tissue, 0 to about 700 millimoles per kilogram of solvent in the fixative of an osmotically active substance having the capacity to control osmotically induced cell volume changes, 0 to about 0.2 mole per liter of fixative of a mordant having the capacity to enhance staining characteristics, and water, the alkanols having a combined concentration of about 200 to about 800 milliliters per liter of fixative, the diols and triols having a combined concentration of about 150 to about 700 milliliters per liter of fixative, and the acids having a combined concentration of about 0.01 to about 0.5 mole per liter of fixative.
2.
The histological fixative of claim 1 in which an effective amount of the osmotically active substance is added and is at least one member selected from the group consisting of a salt, a monosaccharide, a disaccharide, and a water soluble polymer.
The histological fixative of claim 1 in which an effective amount of the osmotically active substance is added and is at least one member selected from the group consisting of a salt, a monosaccharide, a disaccharide, and a water soluble polymer.
3.
The histological fixative of claim 1 in which an effective amount of the mordant is added and is at least one member selected from the group consisting of a salt of a metal ion having an oxidation state of at least two, picric acid, and an alkali dichromate.
The histological fixative of claim 1 in which an effective amount of the mordant is added and is at least one member selected from the group consisting of a salt of a metal ion having an oxidation state of at least two, picric acid, and an alkali dichromate.
4.
The histological fixative of claim 2 in which an effective amount of the mordant is added and is at least one member selected from the group consisting of a salt of a metal ion having an oxidation state of at least two, picric acid, and an alkali dichromate.
The histological fixative of claim 2 in which an effective amount of the mordant is added and is at least one member selected from the group consisting of a salt of a metal ion having an oxidation state of at least two, picric acid, and an alkali dichromate.
5.
The histological fixative of claim 1 in which said alkanol is at least one member selected from the group consisting of methanol and ethanol; the diol and triol is at least one member selected from the group consisting of ethylene glycol, glycerol, propylene glycol and trimethylene glycol; and said acid is at least one member selected from the group consisting of former acid, acetic acid and perchloric acid.
The histological fixative of claim 1 in which said alkanol is at least one member selected from the group consisting of methanol and ethanol; the diol and triol is at least one member selected from the group consisting of ethylene glycol, glycerol, propylene glycol and trimethylene glycol; and said acid is at least one member selected from the group consisting of former acid, acetic acid and perchloric acid.
6.
The histological fixative of claim 2 in which the osmotically active substance is at least one member selected from the group consisting of sodium chloride and zinc chloride, glucose, sucrose, dextran and polyvinylpyrrolidone.
The histological fixative of claim 2 in which the osmotically active substance is at least one member selected from the group consisting of sodium chloride and zinc chloride, glucose, sucrose, dextran and polyvinylpyrrolidone.
7.
The histological fixative of claim 3 in which the mordant is at least one member selected from the group consisting of zinc chloride, strontium chloride, calcium chloride, barium chloride and chromic chloride.
The histological fixative of claim 3 in which the mordant is at least one member selected from the group consisting of zinc chloride, strontium chloride, calcium chloride, barium chloride and chromic chloride.
8.
The histological fixative of claim 4 in which the osmotically active substance is at least one member selected from the group consisting of sodium chloride, zinc chloride, glucose, sucrose, dextran and polyvinylpyrrolidone, and the mordant is at least one member selected from the group consisting of zinc chloride, strontium chloride, calcium chloride, barium chloride and chromic chloride.
The histological fixative of claim 4 in which the osmotically active substance is at least one member selected from the group consisting of sodium chloride, zinc chloride, glucose, sucrose, dextran and polyvinylpyrrolidone, and the mordant is at least one member selected from the group consisting of zinc chloride, strontium chloride, calcium chloride, barium chloride and chromic chloride.
9.
The histological fixative of claim 3 in which said alkanol is ethanol and has a concentration of about 400 milliliters per liter of fixative, said diol and triol is ethylene glycol and has a concentration of about 300 milliliters per liter of fixative, said acid is acetic acid and has a concentration of about 0.17 mole per liter of fixative, and said mordant is zinc chloride and has a concentration of about 5.8 grams per liter of fixative.
The histological fixative of claim 3 in which said alkanol is ethanol and has a concentration of about 400 milliliters per liter of fixative, said diol and triol is ethylene glycol and has a concentration of about 300 milliliters per liter of fixative, said acid is acetic acid and has a concentration of about 0.17 mole per liter of fixative, and said mordant is zinc chloride and has a concentration of about 5.8 grams per liter of fixative.
10.
The histological fixative of claim 4 in which an effective amount of zinc chloride is employed as both the osmotically active substance and the mordant.
The histological fixative of claim 4 in which an effective amount of zinc chloride is employed as both the osmotically active substance and the mordant.
11.
A method for preserving animal tissue which comprises treating said tissue with a mercury and formaldehyde free composition comprising an animal tissue histological fixative amount of a solution comprised of one or more alkanols as a tissue fixative, at least one member selected from the group consisting of an organic diol and triol as a dehydrating agent, at least one acid to precipitate proteins, contribute to nuclear morphological detail, and increase penetration rate of fixative into tissue, 0 to about 700 milliosmoles per kilogram of solvent in the fixative of an osmotically active substance having the capacity to control osmotically induced cell volume changes, 0 to about 0.2 mole per liter of fixative of a mordant having the capacity to enhance staining characteristics and water, the alkanols having a combined concentration of about 200 to about 800 milliliters per liter of fixative, the diols and triols having a combined concentration of about 150 to about 700 milliliters per liter of fixative, and the acids having a combined concentration of about 0.01 to about 0.5 mole per liter of fixative.
A method for preserving animal tissue which comprises treating said tissue with a mercury and formaldehyde free composition comprising an animal tissue histological fixative amount of a solution comprised of one or more alkanols as a tissue fixative, at least one member selected from the group consisting of an organic diol and triol as a dehydrating agent, at least one acid to precipitate proteins, contribute to nuclear morphological detail, and increase penetration rate of fixative into tissue, 0 to about 700 milliosmoles per kilogram of solvent in the fixative of an osmotically active substance having the capacity to control osmotically induced cell volume changes, 0 to about 0.2 mole per liter of fixative of a mordant having the capacity to enhance staining characteristics and water, the alkanols having a combined concentration of about 200 to about 800 milliliters per liter of fixative, the diols and triols having a combined concentration of about 150 to about 700 milliliters per liter of fixative, and the acids having a combined concentration of about 0.01 to about 0.5 mole per liter of fixative.
12.
The method of claim 11 in which an effective amount of the osmotically active substance is added and is at least one member selected from the group consisting of a salt, a monosaccharide, a disaccharide, and a water soluble polymer.
The method of claim 11 in which an effective amount of the osmotically active substance is added and is at least one member selected from the group consisting of a salt, a monosaccharide, a disaccharide, and a water soluble polymer.
13.
The method of claim 11 in which an effective amount of the mordant is added and is at least one member selected from the group consisting of a salt of a metal ion having an oxidation state of at least two, picric acid, and an alkali dichromate.
The method of claim 11 in which an effective amount of the mordant is added and is at least one member selected from the group consisting of a salt of a metal ion having an oxidation state of at least two, picric acid, and an alkali dichromate.
14.
The method of claim 12 in which an effective amount of the mordant is added and is at least one member selected from the group consisting of a salt of a metal ion having an oxidation state of at least two, picric acid and an alkali dichromate.
The method of claim 12 in which an effective amount of the mordant is added and is at least one member selected from the group consisting of a salt of a metal ion having an oxidation state of at least two, picric acid and an alkali dichromate.
15.
The method of claim 11 in which the alkanol is at least one member selected from the group consisting of methanol and ethanol; the diol and triol is at least one member selected from the group consisting of ethylene glycol, glycerol, propylene glycol and trimethylene glycol; and said acid is at least one member selected from the group consisting of formic acid, acetic acid and perchloric acid.
The method of claim 11 in which the alkanol is at least one member selected from the group consisting of methanol and ethanol; the diol and triol is at least one member selected from the group consisting of ethylene glycol, glycerol, propylene glycol and trimethylene glycol; and said acid is at least one member selected from the group consisting of formic acid, acetic acid and perchloric acid.
16.
The method of claim 12 in which the salt is at least one member selected from the group consisting of sodium chloride and zinc chloride; the monosaccharide is glucose; the disaccharide is sucrose; and the polymer is at least one member selected from the group consisting of dextran and polyvinylpyrrolidone.
The method of claim 12 in which the salt is at least one member selected from the group consisting of sodium chloride and zinc chloride; the monosaccharide is glucose; the disaccharide is sucrose; and the polymer is at least one member selected from the group consisting of dextran and polyvinylpyrrolidone.
17.
The method of claim 13 in which the salt is at least one member selected from the group consisting of zinc chloride, strontium chloride, calcium chloride, barium chloride and chromic chloride.
The method of claim 13 in which the salt is at least one member selected from the group consisting of zinc chloride, strontium chloride, calcium chloride, barium chloride and chromic chloride.
18.
The method of claim 14 in which the salt is at least one member selected from the group consisting of zinc chloride, strontium chloride, calcium chloride, barium chloride and chromic chloride.
The method of claim 14 in which the salt is at least one member selected from the group consisting of zinc chloride, strontium chloride, calcium chloride, barium chloride and chromic chloride.
19.
The method of claim 11 in which said alkanol is ethanol and has a concentration of about 400 milliliters per liter of fixative, said diol and triol is ethylene glycol and has a concentration of about 300 milliliters per liter of fixative, said acid is acetic acid and has a concentration of about 0.17 mole per liter of fixative and said mordant is zinc chloride and has a concentration of about 5.8 grams per liter of fixative.
The method of claim 11 in which said alkanol is ethanol and has a concentration of about 400 milliliters per liter of fixative, said diol and triol is ethylene glycol and has a concentration of about 300 milliliters per liter of fixative, said acid is acetic acid and has a concentration of about 0.17 mole per liter of fixative and said mordant is zinc chloride and has a concentration of about 5.8 grams per liter of fixative.
20.
The method of claim 14 in which an effective amount of zinc chloride is employed as both the osmotically active substance and the mordant.
The method of claim 14 in which an effective amount of zinc chloride is employed as both the osmotically active substance and the mordant.
21.
The method of claim 11 in which the animal tissue is prepared for microscopic examination by cutting a section of the animal tissue of suitable size for said examination and the aforesaid treating is effected upon said section of animal tissue.
The method of claim 11 in which the animal tissue is prepared for microscopic examination by cutting a section of the animal tissue of suitable size for said examination and the aforesaid treating is effected upon said section of animal tissue.
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US07/107,026 US4946669A (en) | 1987-10-09 | 1987-10-09 | Histological fixatives |
US107,026 | 1987-10-09 |
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EP1654361B2 (en) | 2003-07-25 | 2023-01-18 | Life Technologies Corporation | Methods and compositions for preparing rna from a fixed sample |
CA2543782A1 (en) * | 2003-10-23 | 2005-05-06 | Georgetown University | Method for two- and three-dimensional microassembly of patterns and structures |
KR20060115366A (en) | 2003-10-24 | 2006-11-08 | 더 유니버시티 오브 마이애미 | Simplified tissue processing |
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EP1804045B1 (en) * | 2005-12-30 | 2014-03-12 | QIAGEN GmbH | Method and Kit for treating a biological sample |
WO2007084429A2 (en) * | 2006-01-13 | 2007-07-26 | Ventana Medical Systems, Inc. | Biological sample processing composition and method |
EP1965190A1 (en) * | 2007-02-27 | 2008-09-03 | Qiagen GmbH | Fixation of a biological sample |
EP2027922A1 (en) | 2007-08-02 | 2009-02-25 | Qiagen GmbH | Method and device for securing/stabilising a sample |
US8338130B2 (en) * | 2011-02-25 | 2012-12-25 | Medical Chemical Corporation | Universal fecal fixative comprising a low molecular weight alcohol, a zinc salt and an organic acid |
JP6757670B2 (en) * | 2014-05-28 | 2020-09-23 | キアゲン ゲーエムベーハー | Fixing composition for cell-containing liquid samples |
US11313772B2 (en) | 2015-11-13 | 2022-04-26 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Fixatives and methods of use |
JP6721899B2 (en) * | 2016-03-30 | 2020-07-15 | 国立大学法人 大分大学 | Embedding method for electron microscope mainly composed of highly hydrophilic polymer monomer |
CN107802889B (en) * | 2017-12-08 | 2019-11-01 | 上海微创心通医疗科技有限公司 | A kind of animal derived collagenous tissue material of dry state and preparation method thereof |
CN116670497A (en) * | 2020-11-11 | 2023-08-29 | 乔治·梅森研究基金会股份有限公司 | Laser capture microdissection visualization chemistry |
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FR1017026A (en) * | 1950-05-03 | 1952-11-28 | Technicon Chemical Company | Liquid for the treatment of histological tissues |
US3546334A (en) * | 1965-05-21 | 1970-12-08 | Lerner Lab Inc | Composition for fixing and protecting a smear of body cells and method of applying same |
JPS5428628B2 (en) * | 1972-05-27 | 1979-09-18 | ||
US3862300A (en) * | 1973-03-07 | 1975-01-21 | Applied Bioscience | Histological fixative |
US3997656A (en) * | 1973-06-25 | 1976-12-14 | Applied Bioscience | Tissue staining method and composition |
US4328256A (en) * | 1978-08-18 | 1982-05-04 | Queen's University At Kingston | Preservation of green plant tissues |
CA1103475A (en) * | 1978-08-18 | 1981-06-23 | Cesar Romero-Sierra | Preservation of green plant tissues |
CA1103476A (en) * | 1979-01-30 | 1981-06-23 | Cesar Romero-Sierra | Preservation of douglas fir needles |
US4486416A (en) * | 1981-03-02 | 1984-12-04 | Soll David B | Protection of human and animal cells subject to exposure to trauma |
US4404181A (en) * | 1981-10-15 | 1983-09-13 | Cambridge Chemical Products, Inc. | Extended-life tissue fixative composition and method of using the same |
US4493821A (en) * | 1982-02-04 | 1985-01-15 | Harrison James S | Preservative and fixative preparations for biological systems |
DE3238639A1 (en) * | 1982-10-19 | 1984-04-19 | Karlheinz 7900 Ulm Bachhuber | METHOD FOR PRODUCING THIN SECTIONS OF BIOLOGICAL TISSUES AND CURABLE INFILTRATION AGENT FOR SUCH TISSUES |
JPS6130501A (en) * | 1984-07-20 | 1986-02-12 | Kanegafuchi Chem Ind Co Ltd | Method for preserving living body |
-
1987
- 1987-10-09 US US07/107,026 patent/US4946669A/en not_active Expired - Lifetime
-
1988
- 1988-10-04 IE IE300488A patent/IE62265B1/en not_active IP Right Cessation
- 1988-10-05 ES ES88116454T patent/ES2051810T3/en not_active Expired - Lifetime
- 1988-10-05 EP EP88116454A patent/EP0311035B1/en not_active Expired
- 1988-10-05 JP JP63251741A patent/JPH076893B2/en not_active Expired - Lifetime
- 1988-10-05 DE DE8888116454T patent/DE3873675T2/en not_active Expired - Fee Related
- 1988-10-07 DK DK562288A patent/DK167134B1/en not_active IP Right Cessation
- 1988-10-11 CA CA000579772A patent/CA1323550C/en not_active Expired - Fee Related
-
1992
- 1992-08-27 GR GR920401881T patent/GR3005550T3/el unknown
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DE3873675D1 (en) | 1992-09-17 |
JPH01199160A (en) | 1989-08-10 |
EP0311035A3 (en) | 1990-01-10 |
JPH076893B2 (en) | 1995-01-30 |
US4946669A (en) | 1990-08-07 |
IE62265B1 (en) | 1995-01-11 |
DK562288A (en) | 1989-04-10 |
GR3005550T3 (en) | 1993-06-07 |
ES2051810T3 (en) | 1994-07-01 |
IE883004L (en) | 1989-04-09 |
DE3873675T2 (en) | 1992-12-24 |
DK562288D0 (en) | 1988-10-07 |
DK167134B1 (en) | 1993-09-06 |
EP0311035B1 (en) | 1992-08-12 |
EP0311035A2 (en) | 1989-04-12 |
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