CN103235137A - Method for detecting nucleoproteins of spermatogenic cells through utilizing indirect immunofluorescent staining - Google Patents

Abstract

A method for detecting the nucleoproteins of spermatogenic cells through utilizing indirect immunofluorescent staining comprises the following steps: separating convoluted seminiferous tubules from the testis tissues of an animal, utilizing a hypotonic solution to process spermatogenic cells, and detecting the nucleoproteins of the spermatogenic cells through adopting indirect immunofluorescent staining, wherein the hypotonic solution comprises 17mM of sodium citrate, 50mM of sucrose, 5mM of EDTA, 0.5mM of DTT, 0.5mM of PMSF and 30mM of Tris and having a pH value of 8.2. The method uses the hypotonic solution to carry out permeable treatment of the cells in order to rapidly expand the cells, disperse chromatins and fully release the proteins in cell nuclei, so the spermatogenic cells can well combine with primary antibodies, thereby the combination efficiencies of secondary antibodies are enhanced, fluorescent staining is complete and full, and the preparation of spermatogenic cell chromosomes and karyotyping are successfully carried out.

Description

Utilize indirect IF staining to detect the method for androgone nucleoprotein

Technical field

The present invention relates to a kind of biochemistry detection method, particularly relate to a kind of detection method of boar androgone nucleoprotein.

Background technology

The incidence of disease of sterility is up to 15%～20% at present, and wherein bridegroom's or husband's side factor accounts for 1/2nd.Male sterility is related to commonwealth quality and following population and develops in a healthy way as the subject matter of male reproductive health, has become the focal issue that presses for social concerns and need to be resolved hurrily.Carry out the male reproductive health fundamental research, the molecular mechanism that discloses spermatogenesis and maturation can provide the laboratory foundation for the pathogenesis of illustrating male sterility, helps the development of clinical male sterility diagnostic techniques.

Mammalian sperm be a complexity and special cell differentiation procedure is divided into m period, meiophase and abnormal shaping phase three phases.Wherein, it is two uniquenesses and the key link that the metamorphosis of meiosis and spermatoblast is shaped, and its special cell differentiation mode is that other any cell is not available in the body, wherein must contain some special mechanism interior.If related gene defective, sudden change or abnormal expression all may make the sperm dyspoiesis, cause male sterility.Find and analyze the expression characteristic of this genoid, can provide the Study on Value clue for the molecular mechanism of further understanding sucking sperm generation.

Gene is the carrier of hereditary information, and protein is only the final executor of cell function.Therefore, the gene functional research of genome times afterwards comprehensively mainly concentrates on protein level, and the accurate location of clear and definite protein in histocyte is the necessary basis of research protein function.The androgone that spermatogenesis process different phase occurs comes in every shape, the function difference, remarkable change also can take place in its nuclear form, can replace histone gradually as nucleoprotamine, combines closely with chromatin, the chromatin height is concentrated, and nuclear volume just can significantly dwindle; Tubulin is synthetic in a large number, and cell guiding nuclear becomes microscler from circle, just can possess the grown form of mature sperm.These variations all need a series of new protein moleculars constantly to synthesize and the performance normal function could be finished smoothly.In order to further investigate kind and the function of androgone nuclear protein matter, at first need the Subcellular Localization of clear and definite protein.Endonuclear protein has the duplicate protection of cell membrane and nuclear membrane and with nuclear chromatin close relationship is arranged and be present in; must guarantee that when carrying out protein positioning research antibody molecule can combine closely with target protein, avoid chromatin to the interference of target protein and antibody simultaneously.

At present, most researchers all adopt the method for immunohistochemistry or indirect IF staining that protein is positioned research.Obviously can distinguish because the fluorescent dye result is in bright gay color, the positive is painted, can carry out simultaneously the common location of multiple proteins by several different fluorescence, can also utilize laser co-focusing to carry out image stack and stereo-picture analysis, make the indirect IF staining technology in the work of research protein positioning, have incomparable advantage.But want to obtain desirable fluorescent dye result, also note and use appropriate methods and material.

At first, primary antibodie and fluorescence labeling two that immunofluorescence dyeing need possess high specificity resist, and secondly, the preparation of specimen material and processing also are the experiment key of success.If the target protein of research is positioned at nucleus, to guarantee that also antibody can pass cell membrane and nuclear membrane enters nucleus, with the abundant combination of protein in the nuclear.In order to improve the permeability of cell membrane and nuclear membrane antagonist, need to use chemical reagent that cell is carried out penetrating processing.At present, general experiment all handles to increase biomembranous penetrating ability with TitonX-100.Triton X-100 chemical name is Triton X-100, it is a kind of detergent, can with biological membrane in phospholipids incorporate form soluble complex, also can be combined with the hydrophobic region of memebrane protein and form compound in its hydrophobic side, therefore can make phospholipid bilayer loose, the permeability of big molecular antibody is strengthened, in immunohistochemistry and immunocytochemistry often as penetrating dose of cell.But the biological membrane of the TitonX-100 of high concentration has bigger destructiveness, and can make cell and inner organelle dissolving thereof, might cause losing of nuclear protein matter.

Summary of the invention

The objective of the invention is to overcome the defective of prior art, providing a kind of is material with the testis tissue, utilizes hypotonic medium to prepare androgone, detects the method for androgone nucleoprotein by indirect IF staining.

The method that the present invention utilizes indirect IF staining to detect androgone nucleoprotein is to separate convoluted seminiferous tubule from the testis tissue of animal, utilizes hypotonic medium to handle androgone, adopts the indirect IF staining method to detect androgone nucleoprotein.

Wherein, include 17mM sodium citrate, 50mM sucrose, 5mM EDTA, 0.5mM DTT, 0.5mM PMSF in the described hypotonic medium, 30mM Tris, the pH value of hypotonic medium is 8.2.

The method that the present invention utilizes indirect IF staining to detect androgone nucleoprotein specifically may further comprise the steps:

1) gets the animal testis tissue of the PBS solution that places precooling, peel off the tunica albuginea of testis tissue, convoluted seminiferous tubule is fully separated, fully wash convoluted seminiferous tubule with the PBS solution of precooling;

2) cover convoluted seminiferous tubule with hypotonic medium, handle 10min for 37 ℃;

3) draw celliferous hypotonic medium and be added in advance on the microslide that lysine is handled, after room temperature is dried, 1%BSA room temperature sealing 30min;

4) drip primary antibodie, place 2h in 37 ℃ of wet boxes, PBS washing 3 times, each 5min;

5) drip two and resist, place 1h in 37 ℃ of wet boxes, PBS washing 3 times, each 5min;

6) DAPI redyes 1min, PBS washing 3 times, each 5min;

7) the anti-mountant mounting that fades, observations under the fluorescent microscope is taken the photograph sheet.

Hypotonic medium is the solution that a kind of osmotic pressure and ionic strength all are lower than the normal cell physiological condition, rely on counter osmosis, when cell is in hypotonic environment, moisture can enter cell rapidly, make its expansion, even break, can make simultaneously to adhere to chromosomal material and scatter, fully show chromosomal form.Common hypotonic medium principal ingredient has 4.4g/L sodium citrate, 0.65M potassium glycerinophosphate, 0.075M potassium chloride etc.

The present invention is by above-mentioned hypotonic principle, make a kind of hypotonic medium of improvement, handle androgone with it, cell is expanded naturally, chromosome is sprawled, and the nucleoprotein of being combined with chromatin is loosened, and fully exposes antigen site, enter in the nucleus and combine closely with target protein thereby be conducive to antibody molecule, strengthen fluorescence developing.This is the important evidence that the inventive method is implemented, and hypotonic effect depends primarily on chemical composition and the processing time of hypotonic medium.

In the constituent of hypotonic medium of the present invention, sodium citrate is a kind of weak acid strong alkali salt, can form stronger pH buffering agent with the citric acid compatibility, can keep cell for hypotonic medium and be in the optimum pH buffer system; Secondly, all right chelating bivalent cation of sodium citrate is connected with certain dissociation to the cell between the cell monolayer, can play the purpose of disintegrated tissue.Sucrose is a kind of natural component, and is nontoxic, has well water-soluble and osmosis.EDTA is a kind of calcium chelating agent, is used for hypotonic medium and can promotes cell membrane to the interference of the penetrating of material and eliminating metallic ion.DTT has antioxidation, is commonly used for reductive agent; One of purposes of adding DTT in hypotonic medium is to stablize chromatin, reduces the gathering between the chromatin; In addition, DTT also effectively in the protected protein matter molecule disulfide bond be in reducing condition, stop owing to form disulfide bond between the halfcystine and cause the protein molecule space structure to change.Bibliographical information is arranged, and DTT can be dissolved out nucleus albumen under appropriate condition from cyto-chromatin, utilizes DTT to protect chromatin Structure well in the inventive method and promotes the release of nucleoprotein.PMSF is a kind of protease inhibitors, and effectively the Profilin enzyme is kept the integrality of protein structure to the degradation of albumen.Tris is commonly used for biological buffer, and in the environment of pH8.2, the Tris damping fluid is weakly alkaline solution, helps DNA and protein deprotonation, promotes dissociating of the two.Add EDTA in the Tris damping fluid and also be usually used in stablizing of DNA and storage.The physiological action of above-mentioned various compositions also is the important evidence that the inventive method is implemented.

The present invention uses hypotonic medium that cell is carried out penetrating processing, make the cell rapid expanding, chromatin scatters, nuclear protein matter fully discharges, androgone can be combined with primary antibodie better, then strengthen two anti-joint efficiencies, make fluorescent color more cmpletely, thereby can successfully prepare androgone chromosome and carry out karyotyping.Simultaneously, the constituent safety non-toxic of hypotonic medium of the present invention can protect nuclear protein matter to keep original structure well.

Embodiment

Below be example with the mouse, describe the present invention in detail and utilize indirect IF staining to detect the operation steps of androgone nucleoprotein method.But the inventive method also not only is confined to mouse, and the androgone of other boars can utilize the inventive method to carry out the indirect IF staining of nucleoprotein equally.

1. disconnected neck is put to death the suitably male mice in age in week, 75% alcohol immersion, and the mouse testis tissue is taken out in sterile working, places the plate that contains precooling PBS solution.

2. carefully peel off tunica albuginea, reject blood vessel as far as possible, convoluted seminiferous tubule is fully separated, move in another plate that contains precooling PBS solution.

3. with the PBS solution washing for several times, remove other impurity such as organizing fragment.

4. under dissecting microscope, the wall scroll convoluted seminiferous tubule that will separate with meticulous sharp mouth tweezers stretches.

5. the hypotonic medium of getting fresh configuration covers convoluted seminiferous tubule in right amount, 37 ℃ of penetrating processing 10 minutes.

6. draw the liquid after an amount of the processing, be added in advance on the microslide that lysine is handled, room temperature is dried.

6. the sealing of 1%BSA room temperature is 30 minutes.

7. dropping primary antibodie was placed 2 hours in 37 ℃ of wet boxes.

8. the PBS washing is 3 times, each 5 minutes.

9. drip two and resist, placed 1 hour in 37 ℃ of wet boxes.

10. the PBS washing is 3 times, each 5 minutes.

11. DAPI redyed 1 minute.

12. PBS washing 3 times, each 5 minutes.

13. the anti-mountant mounting that fades.

14. observations under the fluorescent microscope is taken the photograph sheet.

Claims (2)

1. utilize indirect IF staining to detect the method for androgone nucleoprotein, it is characterized in that from the testis tissue of animal, separating convoluted seminiferous tubule, utilize hypotonic medium to handle androgone, adopt the indirect IF staining method to detect androgone nucleoprotein, wherein, include 17mM sodium citrate, 50mM sucrose, 5mM EDTA, 0.5mM DTT, 0.5mM PMSF in the described hypotonic medium, 30mM Tris, the pH value of hypotonic medium is 8.2.

2. the method for utilizing indirect IF staining to detect androgone nucleoprotein according to claim 1 is characterized in that may further comprise the steps:

1) gets the animal testis tissue of the PBS solution that places precooling, peel off the tunica albuginea of testis tissue, convoluted seminiferous tubule is fully separated, fully wash convoluted seminiferous tubule with the PBS solution of precooling;

2) cover convoluted seminiferous tubule with hypotonic medium, handle 10min for 37 ℃;

3) draw celliferous hypotonic medium and be added in advance on the microslide that lysine is handled, after room temperature is dried, 1%BSA room temperature sealing 30min;

4) drip primary antibodie, place 2h in 37 ℃ of wet boxes, PBS washing 3 times, each 5min;

5) drip two and resist, place 1h in 37 ℃ of wet boxes, PBS washing 3 times, each 5min;

6) DAPI redyes 1min, PBS washing 3 times, each 5min;

7) the anti-mountant mounting that fades, observations under the fluorescent microscope is taken the photograph sheet.