CN116952690A - Testis tissue pathology preparation method - Google Patents
Testis tissue pathology preparation method Download PDFInfo
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
- G01N2001/302—Stain compositions
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- 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
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Abstract
The invention discloses a testis tissue pathology preparation method, and relates to the field of pathology tissue preparation; firstly, a dense fibrous tissue envelope on the surface of testis tissue adopts a method of punching holes at two ends so as to achieve the perfect fixing effect of the fixing liquid which is quickly immersed into the tissue, the morphology of a section seminiferous tubule is regular, the layers of the seminiferous cells at each development stage are clear, the cell structure is clear, the chromatin is clear and clear, and the observation effects of euchromatin and heterochromatin can be distinguished; the whole testis tissue is fixed for 8-12 hours through mDF, so that the testis tissue is complete in shape, seminiferous tubules are precisely arranged, the tubules are round, the tissue cell shape is clear, the cell classification is obvious, the morphological detail is good, and the testis morphology and structure are complete after HE dyeing; after PAS glycogen is dyed, the testicle top body is positioned clearly and is well colored; IHC experiments showed good antigen preservation.
Description
Technical Field
The invention relates to the field of pathological tissue preparation, in particular to a testicle tissue pathological preparation method.
Background
The incidence of male infertility is in an increasing trend worldwide, and can be further divided into azoospermia, oligospermia, asthenospermia and normal sperm count infertility according to semen analysis results; testis disease such as testis tumor, testis tuberculosis, testis syphilis, testis nonspecific inflammation and the like can cause spermatogenic dysfunction, basic research is required for the pathogenesis reasons, and especially, observation and research of tissue sections under a microscope are required;
as a part of clinical work, the related diseases of male sterility need to be researched for occurrence, development and treatment of the diseases, pathological features of the diseases and related signal molecular mechanisms are researched, and the selected animal model is a C57 mouse, so that the animal model has the advantages of small size, rapid propagation, stable strain, high result precision, rich experimental research data and strong reference contrast. And the genome of the C57 mouse is highly homologous with the human genome, and 99% of genes of the C57 mouse can find homologous genes in the human genome;
therefore, the research of the C57 mouse testicle pathological film-making method has important significance for the research of the pathogenesis of male reproductive system diseases; high quality tissue sections are required for conventional pathological morphology observation, molecular pathology RNA-scope detection and related research of immunofluorescence and immunohistochemistry at protein level, and the tissue sections refer to pathological sections prepared by taking biological tissues as detection materials through a series of methods, and pathological morphology changes of the tissues and cells are observed under a microscope after staining; the tissue slice with high quality is made, firstly, proper fixing liquid and fixing time are needed to be selected to support the follow-up work, the integrity of tissues and cell structures can be affected by the selection of the fixing liquid and the inappropriateness of the fixing time course, and testis tissues have specificity, soft, tender and crisp quality, so that perfect slicing and dyeing are important in the study;
the testicle histopathological morphology evaluation is an important component for researching the reproductive pathology and the growth and development of experimental animals; the seminiferous epithelial cells of testis tissue have lower protein content and are more difficult to fix than other organs and tissues; testis is composed of substance and matrix, and irregular arrangement, structural deficiency, disorder of matrix, shrinkage of cell nucleus, tissue flaking during dyeing and other problems caused by inadequate fixing solution and fixing time are unfavorable for the promotion of subsequent work.
In summary, the current testis has the following technical problems in the tissue slicing process: because the testis is wrapped by dense and tough fibrous tissues, the interior is soft and fragile (such as jellied bean curd), the slicing is difficult to manufacture, 10% formaldehyde fixing liquid is routinely used in clinical testis biopsy and animal experiments, the 10% formaldehyde fixing liquid has strong permeability, simple configuration and convenient operation, but the formaldehyde content is higher, under the effect of the strong permeability, the contraction degree of the tissues is obvious, the condition that seminiferous tubules and interstitials are seriously contracted occurs, and the observation of tissue structures and cell morphology is influenced; in the process of making mouse testis sections, reasonable treatment of tissues is required to ensure the integrity of tissue structures. Multiple stains are often required in research work, and matching of different fixatives to different stains is critical. The prior art generally lacks standardization according to experimenters experience and using habit for fixing tissues and optionally selecting fixing liquid, so that perfect pathological sections are difficult to manufacture, and pathological observation of tissue cells under different staining is seriously affected.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a preparation method of testicle tissue pathology.
In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:
a preparation method of testis tissue pathology comprises the following steps
S1: pretreating the testes of the C57 mice; c57 mouse testes are obtained, testers group the testes, each group of 12 testes respectively carry out comparison experiments of different fixing solutions and different fixing times, different staining is adopted, and the optimal effect of the three fixing solutions in different staining is explored;
s2: hole punching treatment is carried out on two ends of the testis; different fixation pretreatment methods such as middle cutting, hole punching by a syringe at two ends, complete fixation and the like are respectively adopted for testis tissues in the early stage of experiments; the middle incision mode cannot keep the complete structural form after incision due to the characteristics of tissue in testis, the tissue cannot be crushed in the incision process, slices which are beneficial to observation cannot be obtained even if the tissue is fixed, the subsequent fixing liquid is not needed to be fixed, and the comparison result of the other two treatment modes before fixing shows that the two end hole punching treatment methods have the best fixing effect, the shape of the seminiferous tubules of testis is regular and closely arranged, the layers of seminiferous cells are distinct, and the cell structure is clear; in the complete fixation method, the testis center cannot be well fixed due to the blocking of the compact fibrous tissue envelope toughness of the outer layer of the testis, so that part of cells are subjected to autolysis change, and pathological observation is seriously influenced; the subsequent experiments adopt a method of punching holes at two ends to treat tissues;
s3: placing testis into fixing liquid; grouping according to three fixing solutions and different fixing times, respectively placing the three fixing solutions in 10% NBF, mDF and BF, respectively fixing each fixing solution for 8h, 12h,24h and 48h to obtain different fixing solutions and fixed testes in different states in different times, respectively carrying out different staining to obtain sections with different effects, and observing and comparing under a microscope;
s4: post-treating testes; gradient alcohol dehydration is carried out on the testis after fixation: overnight with 75% ethanol, with 85% ethanol for 4h, with 95% ethanol for 1.5h, with anhydrous ethanol for 1h, with xylene for 20min, and with wax for 2h, and making into wax block;
s5: dyeing testes; staining each set of different time samples using hematoxylin-eosin staining (HE), glycogen staining (PAS), immunohistochemical staining (IHC);
further, the preparation method of the testis tissue pathology further comprises the following steps:
s3.1: mDF (modified Davidson's fluid, modified Dai Wensen fixative) for 8-12h; according to absolute ethyl alcohol, formaldehyde stock solution, glacial acetic acid and distilled water according to 3:6:1:10, preparing the mixture in proportion;
s5.1: HE (hematoxylin-eosin) staining; dewaxing slices in xylene to water after 40min in a 60-degree oven, dehydrating with gradient alcohol after conventional hematoxylin and eosin staining, and sealing with neutral resin;
further, the preparation method of the testis tissue pathology further comprises the following steps of
S3.2: bouins is fixed for 12h; saturated picric acid solution (1.22%), formaldehyde stock solution and glacial acetic acid according to 15:5:1, mixing in proportion;
s5.2: PAS (glycogen) staining; dewaxing slices in a 60-DEG oven for 40min, washing with distilled water for 1-2min, oxidizing with periodic acid for 30min, washing with distilled water, removing excessive water, washing with flowing water for 30min in schiff dye liquor, dyeing with hematoxylin, washing with flowing water, dehydrating with gradient alcohol, and sealing with neutral resin;
further, the preparation method of the testis tissue pathology further comprises the following steps of
S3.3:10% neutral formalin (NBF) solution was fixed for 8h; according to the formaldehyde stock solution, PBS and distilled water according to the following ratio of 1:4.5:4.5, preparing in proportion;
s5.3: IHC (Immunohistochemistry) staining; dewaxing slices in xylene to water after 40min in a 60-DEG oven, and repairing citrate buffer solution with microwave antigen; PBS is used for 3 times, endogenous peroxidase blocking agent is placed for 30min at room temperature, PBS is used for 3 times, goat serum is blocked for 40min, primary antibody is used for 4 degrees overnight, PBS is used for 6 times, secondary antibody is placed for 40min at 37 degrees, PBS is used for 6 times, and DAB color development is performed. Hematoxylin counterstain cell nuclei, gradient alcohol dehydration and neutral resin sealing;
further, the preparation method of the testis tissue pathology further comprises the following steps of
S3.4: mDF for 12h; according to absolute ethyl alcohol, formaldehyde stock solution, glacial acetic acid and distilled water according to 3:6:1:10, preparing the mixture in proportion;
s5.4: PAS dyeing; dewaxing slices in a 60-DEG oven for 40min, washing with distilled water for 1-2min, oxidizing with periodic acid for 30min, washing with distilled water, removing excessive water, washing with flowing water for 30min in schiff dye liquor, dyeing with hematoxylin, washing with flowing water, dehydrating with gradient alcohol, and sealing with neutral resin;
further, the preparation method of the testis tissue pathology further comprises the following steps of
S3.5: mDF for 12h; according to absolute ethyl alcohol, formaldehyde stock solution, glacial acetic acid and distilled water according to 3:6:1:10, preparing the mixture in proportion;
s5.5: IHC staining; dewaxing slices in xylene to water after 40min in a 60-DEG oven, and repairing citrate buffer solution with microwave antigen; PBS is used for 3 times, endogenous peroxidase blocking agent is placed for 30min at room temperature, PBS is used for 3 times, goat serum is blocked for 40min, primary antibody is used for 4 degrees overnight, PBS is used for 6 times, secondary antibody is placed for 40min at 37 degrees, PBS is used for 6 times, and DAB color development is performed. Hematoxylin counterstain cell nuclei, gradient alcohol dehydration and neutral resin sealing;
the invention has the beneficial effects that:
a testis tissue pathology preparation method, through adopting the method of the thin needle punching of both ends of tissue after drawing materials of C57 mouse testis, can reach the effect of the fast fixed tissue cell, and the punching is because testis shows to be the dense fibrous tissue to wrap up, make the fixing fluid unable to immerse fast and make the central cell autolyze change; the section seminiferous tubules manufactured after hole punching and fixing are regular in morphology, clear in gradation of seminiferous cells in each development stage, clear in cell structure, clear in chromatin, capable of distinguishing euchromatin, observation effects of heterochromatin and the like;
the whole testis tissue is fixed for 8-12 hours through mDF, so that the testis tissue is complete in shape, seminiferous tubules are precisely arranged, the tubules are round, the tissue cell shape is clear, the cell classification is obvious, the morphological detail is good, and the testis morphology and structure are complete after HE dyeing; after PAS glycogen is dyed, the positioning of the acrosome of spermatid in testis is clear, and the color is good; IHC experiments show that the antigen is well preserved; PAS is fixed for 8-12 hours by using Bouin's solution, so that the dyeing effect of the testicular sperm cell acrosome is clear, the effect is good, the cell nucleus is blue-purple, the morphology of the sperm cells is regular, the purple acrosome can be clearly observed, and the high-quality differentiation sperm producing period can be realized; IHC staining is performed by using 10% neutral formalin solution for 8 hours, so that the preservation effect of antigen is optimal, the positive intensity of the histochemical staining is high, the antigen positioning is accurate, the advantages and disadvantages of three fixing solutions are clarified, and the optimal fixing time is determined; the optimal matching of the three fixing solutions with different dyeings is determined, and the optimal effect is obtained.
Of course, it is not necessary for any of the methods of practicing the present invention to achieve all of the advantages set forth above at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a section of tissue after fixation of two ends of a testis by perforation according to an embodiment of the invention;
FIG. 2 is a section of tissue after fixation of a testicle without puncture according to an embodiment of the present invention;
FIG. 3 is a graph showing comparison of HE staining effects of testis tissue of a C57 mouse after 12h fixation with three different fixatives according to the embodiment of the present invention;
FIG. 4 is a graph showing the comparative effect of PAS staining on testis tissue of a C57 mouse after 12h fixation with three different fixatives according to the example of the present invention;
FIG. 5 is a graph showing comparison of IHC staining effects of testis tissue of a C57 mouse after 12h fixation with three different fixatives according to the embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Pretreatment of C57 mouse testes included;
1. material
1.1 laboratory animals
20C 57 mice (40 testes) with the age of 6-8 weeks are provided by Liaoning province laboratory animal center, and the body mass is 23-25 g.
1.2 preparation of fixative
10% neutral formalin fixation (10% nbf): formaldehyde stock solution, PBS and distilled water are mixed according to the following ratio of 1:4.5: 4.5. Improved Dai Wensen fixative (Motified Davidson Fluid, mDF): absolute ethyl alcohol, formaldehyde stock solution, glacial acetic acid and distilled water according to the following weight ratio of 3:6:1: 10. Bouins fixative (Bouin's Fluid, BF): picric acid saturated solution (1.22%), formaldehyde stock solution and glacial acetic acid according to 15:5:1, and mixing the materials in proportion.
2. Experimental operation
2.1 obtaining materials
After anesthetizing the C57 mice with sodium pentobarbital, the lower abdomen of the C57 mice was opened with an ophthalmic scissors, and bilateral testes of the C57 mice were cut.
Before being put into the fixing liquid, the testis of the C57 mouse is processed by three methods of middle cutting, hole punching by a syringe at two ends and complete fixing.
2.2 fixing
The testis after hole punching was placed in the following three fixatives (10% NBF, mDF, BF) and fixed for 8h, 12h,24h,48h, respectively.
Post-treatment of testes includes;
2.3 Paraffin section preparation
Gradient alcohol dehydration is carried out on the testis after fixation: 75% ethanol overnight, 85% ethanol 4h, 95% ethanol 1.5h, absolute ethanol 1h, xylene transparent for 20min, wax dipping for 2h, and then preparing into wax blocks for standby.
Staining testes including;
2.4 hematoxylin-eosin staining (HE) immunohistochemical staining
The sections were dewaxed to water in xylene after 40min in a 60 degree oven, dehydrated with gradient alcohol after conventional hematoxylin and eosin staining, and observed for morphological structure under microscope after neutral resin encapsulation.
2.5 glycogen staining (PAS)
Dewaxing slices in a 60-DEG oven for 40min, washing with distilled water for 1-2min, oxidizing with periodic acid for 30min, washing with distilled water, removing excessive water, washing with schiff dye liquor for 30min, washing with running water, hematoxylin dyeing, washing with running water, dehydrating with gradient alcohol, and sealing with neutral resin.
2.6 immunohistochemical staining (IHC)
Dewaxing slices in xylene to water after 40min in a 60-DEG oven, and repairing citrate buffer solution with microwave antigen; PBS is used for 3 times, endogenous peroxidase blocking agent is placed for 30min at room temperature, PBS is used for 3 times, goat serum is blocked for 40min, primary antibody is used for 4 degrees overnight, PBS is used for 6 times, secondary antibody is placed for 40min at 37 degrees, PBS is used for 6 times, and DAB color development is performed. Hematoxylin counterstains cell nuclei, gradient alcohol dehydration and neutral resin sealing.
As shown in fig. 1-2, the tissue slice shape is obtained after two ends of the tissue slice shape are fixed in a hole punching mode and an unbunching mode under HE staining, the fixing effect of the hole punching treatment method at the two ends is optimal, the testicle seminiferous tubule shape is regular and compact, the layers of the seminiferous cells are distinct, the cell structure is clear and neat, and the distribution of chromatin in the cells is clear and visible; the middle incision is soft in testis, so that tissues are broken, and subsequent experimental operation cannot be performed; in the complete fixation method, due to the blocking of the ductile structure of the testis outer layer to the penetration of the fixation liquid, the tissue seminiferous tubule cells are slightly autolyzed, and the distribution of chromatin in the cells is unclear, so that the subsequent section experimental observation is affected.
As shown in fig. 1:
the effect exhibited by three fixatives in HE staining:
the tissue testis fixed for 8 hours by 10% NBF has deeper coloration under a low-power microscope, the envelope is seriously wrinkled, the seminiferous tubules are loose in arrangement, larger gaps are visible due to interstitial contraction, and the tubule deformation is seriously obvious. When observed under a high-power microscope, the cell structure of testis is still clear, but the structure level of sperm cells is lost; obvious cell debris can be seen in the seminiferous tubule lumen; the testis morphology is not greatly changed after fixation for 12h,24h and 48 h;
the envelope of testis tissue fixed for 12 hours by Bouis is slightly shrunken under a low-power microscope, seminiferous tubules are closely arranged, seminiferous epithelium and interstitial regions are contracted, types of seminiferous cells can be clearly distinguished under a high-power microscope, and the number of cell fragments in the seminiferous tubules is obviously reduced compared with 10% of NBF fixing liquid; the fixation time is prolonged to 24 hours, and the influence of 48 hours on the testis morphology is not great;
mDF the testis tissue fixed for 12 hours is uniformly colored under a low-power mirror, the envelope is relatively complete, the arrangement of seminiferous tubules is relatively compact, the cytoplasm of the seminiferous tubules is slightly contracted, and the morphology of the tubules is regular; the germ cell structure is clear under the high-power microscope, and the level of the sperm cell is clear; the cell debris quantity in the seminiferous tubule is obviously reduced; a small amount of gaps are reserved between testicle interstitium for 8 hours, slight cracks appear between seminiferous tubules, and the sperm germinal layer is complete and has clear layers; after 24 hours fixation, the testicle interstitial space is slightly larger, the seminiferous tubules are tightly connected, and the sperm germinal layer is slightly unclear in classification; after 48 hours fixation, the testicle interstitial space is increased, even part of the testicle is lost, the structure of part of seminiferous tubules is slightly incomplete, the shape is irregular, and germinal layer cells are slightly unclear;
example 2
As shown in fig. 2
The effect exhibited by three fixatives in PAS staining:
10% NBF is fixed on testis tissue for 8 hours, the whole cell staining is deep, sperm acrosomes in seminiferous tubules are not clear, bouis is fixed on testis cell nuclei for 12 hours to be blue-purple, the sperm structure is clear, purple acrosomes can be seen, the types of seminiferous cells can be clearly separated, mDF is fixed on testis for 12 hours, the types of seminiferous cells are obvious, but the structural definition of the acrosomes is more fuzzy than Bouis;
example 3
As shown in fig. 3
The effect exhibited by three fixatives in IHC staining:
10% NBF is fixed for 8 hours, so that the testis tissue histochemical staining positive intensity and the cell positioning effect are good, the background color is clear, and the nonspecific staining is less; bouis fixes 12 hours testis tissue positive effect is poor, the characteristics of strong middle positive but weak edge positive effect are presented, and serious nonspecific staining occurs; mDF fixed testis cells for 12 hours are clear in coloring and strong in specificity, but the positive intensity is slightly weaker than 10% NBF;
table 1. After the three fixing solutions were fixed for 12 hours, the degrees of the three dyeing effects were compared, the + effect is poor, the ++ effect is moderate, good++ effect
In conclusion, according to the testicle tissue pathology preparation method, the method of puncturing holes at two ends of tissue is adopted rapidly after the C57 mouse testicle is obtained, and the fixation of the testicle center is completed by innovatively matching with the fixing liquid, so that the effects of regular testicle seminiferous tubule morphology, clear cell structure, clear and distinct chromatin and the observation effect of distinguishing euchromatin and heterochromatin are achieved; the whole testis tissue is fixed for 8-12 hours through mDF, so that the testis tissue is complete in shape, seminiferous tubules are precisely arranged, the tubules are round, the tissue cell shape is clear, the cell classification is obvious, the morphological detail is good, and the testis morphology and structure are complete after HE dyeing; after PAS glycogen is dyed, the testicle top body is positioned clearly and is well colored; IHC experiments show that the antigen is well preserved; PAS is fixed for 8-12 hours by using Bouin's solution, so that the dyeing effect of testicle top is clear, glycogen dyeing effect of testicle top is good, cell nuclei are blue-purple, the morphology of sperm cells is regular, purple top can be clearly observed, and the high-quality differential sperm production cycle can be realized; IHC staining is performed by using 10% neutral formalin solution for fixation for 8 hours, so that the preservation effect of the antigen is optimal, the positive intensity of the histochemical staining is high, the antigen positioning is accurate, the advantages and disadvantages of three fixing solutions are clarified, and the optimal fixing time is determined; the innovation defines the best effect of combining three fixing solutions with different dyeings.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (6)
1. A preparation method of testicle tissue pathology is characterized in that: the method comprises the following steps:
s1: pretreating the isolated testis tissue;
s2: punching two ends of the isolated testis tissue;
s3: placing the isolated testis tissue into a fixing solution;
s4: post-treating the isolated testis tissue;
s5: isolated testis tissue was stained.
2. A method of preparing a testicular tissue pathology according to claim 1, wherein: when observing testis tissue morphology, S3: mDF for 8-12h; s5: HE staining.
3. A method of preparing a testicular tissue pathology according to claim 1, wherein: when observing the staining effect of testicular sperm cell acrosome, S3: bouins is fixed for 12h; s5: PAS staining.
4. A method of preparing a testicular tissue pathology according to claim 1, wherein: when preserving antigen, S3:10% neutral formalin (NBF) solution was fixed for 8h; s5: IHC staining.
5. A method of preparing a testicular tissue pathology according to claim 1, wherein: when preserving antigen, S3: mDF for 12h; s5: PAS staining.
6. A method of preparing a testicular tissue pathology according to claim 1, wherein: when observing the staining effect of testicular sperm cell acrosome, S3: mDF for 12h; s5: IHC staining.
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Inventor after: Tian Pengxiang Inventor after: Hao Guimin Inventor after: Feng Tengfei Inventor after: Zhang Yong Inventor before: Tian Pengxiang Inventor before: Hao Guimin Inventor before: Feng Tengfei |