CN115873940A - Biomarker for diagnosing and/or prognostically evaluating azoospermia and application thereof - Google Patents

Abstract

The invention relates to the field of azoospermia diagnosis, in particular to a biomarker for diagnosing and/or prognostically evaluating azoospermia and application thereof, wherein the biomarker is DAZL and SOX9, and HE (hematoxylin-eosin) staining can be further used for determining the type of a non-obstructive azoospermia patient without sperms by detecting the expression level of the DAZL and SOX9, so that a traumatic operation is avoided.

Description

Biomarker for diagnosing and/or prognostically evaluating azoospermia and application thereof

Technical Field

The invention relates to the field of azoospermia diagnosis, in particular to a biomarker for diagnosing and/or prognostically evaluating azoospermia and application thereof.

Background

The incidence rate of male infertility is about 10%. Clinically, male infertility is classified into sexual disorder and normal sexual function, and the latter can be further classified into azoospermia, oligospermia, asthenospermia and sperm number normal sexual infertility according to semen analysis results. Among them, azoospermia accounts for about 15% -20% of male infertility patients, and its causes are various, and can be classified into Obstructive Azoospermia (OA) and non-obstructive azoospermia (NOA).

At present, testicular dissection is one of the main means for accurate diagnosis of azoospermia, but the examination means is complex and long in time, and the influence of traumatic examination on patients is high. Meanwhile, the testicular dissection detection can only judge whether sperms exist in the testis of a patient, and can not judge the severity of the patient without the sperms. The patient cannot be judged to be unable to develop sperm due to a problem in the germ cell development process or to have no germ cells at all.

Disclosure of Invention

Human male germ cells can be classified into spermatogonial stem cells, spermatocytes, haploid sperm cells, and mature sperm depending on the degree of development. Clinically, some patients with azoospermia do not have mature sperm, but germ cells at different developmental stages. This patent carries out the detection of specific marker combination through immunofluorescence to patient's testicle puncture or operation sample, can follow the cell level and accurately assess the developmental degree of germ cell and support cell in the patient's testicle, for clinician provides more accurate judgement, avoids traumatic inspection, can play the guide effect for doctor's treatment in next step. Specifically, the method comprises the following steps:

in a first aspect of the invention, there is provided the use of a biomarker, or a reagent for detecting a biomarker, which is DAZL, in the diagnostic and/or prognostic assessment of azoospermia.

Preferably, the biomarker further comprises SOX9.

Preferably, the reagent for detecting the biomarker is a reagent for detecting the mRNA expression level of the DAZL gene and/or the SOX9 gene, and/or the expression level of the DAZL protein and/or the SOX9 protein.

Further preferably, the detection is the detection of the presence or absence of a biomarker.

Preferably, the test sample is derived from testis or testis puncture extract.

In a second aspect of the invention, there is provided a use of a biomarker, or a reagent for detecting a biomarker, for the manufacture of a product for the diagnostic and/or prognostic assessment of azoospermia, wherein the biomarker is DAZL.

Preferably, the biomarker further comprises SOX9.

Preferably, the reagent for detecting the biomarker is a reagent for detecting the mRNA expression level of the DAZL gene and/or the SOX9 gene, and/or the expression level of the DAZL protein and/or the SOX9 protein.

Further preferably, the detection is the detection of the presence or absence of a biomarker.

Preferably, the test sample is derived from testis or testis puncture extract.

Preferably, the product comprises a diagnostic kit, a gene chip, a microarray or a protein array.

Preferably, the diagnostic kit comprises a reagent for detecting DAZL using an immunofluorescence assay.

Further preferably, the diagnostic kit comprises a reagent for detecting SOX9 using an immunofluorescence method.

Preferably, the reagents used in the immunofluorescence assay include, but are not limited to, blocking solutions (prepared from 5% -15% FBS, 25-35mg/mL BSA and PBS), antigen retrieval buffers (prepared from 0.32-0.40g/L citric acid, 2.5-3.5g/L sodium citrate, preferably, the pH of the antigen retrieval buffer is 6.4), TBST solutions, 1% Triton X-100 solutions, primary and/or secondary reagents.

Preferably, the immunofluorescence method comprises the steps of:

1) Preparation of reagents: respectively preparing confining liquid, antigen repairing buffer liquid, primary antibody and secondary antibody;

2) Baking the slices: taking out the samples, sequentially placing the samples on a dyeing rack, and drying the samples in a 65 ℃ oven for 2 hours;

3) Dewaxing and rehydration: putting the staining rack into a corresponding staining jar for dewaxing and rehydration of paraffin sections:

4) Antigen retrieval: the antigen retrieval buffer (preferably 400-600 ml) is taken in the antigen retrieval cassette and heated to boiling. And then placing the staining rack in an antigen retrieval box, covering the box, continuously heating (preferably heating for 10 min), and taking out the antigen retrieval box, and naturally cold cutting. After cooling to room temperature, placing the staining rack in a staining jar containing TBST solution, and gently shaking and washing on a shaking table (preferably, the washing comprises washing 1-5 times, further preferably washing 3 times, each time for 1-10min, preferably washing 5min each time);

5) Permeability: applying a 1% Triton X-100 solution drop-wise to the tissue section, permeabilizing the tissue section at room temperature, preferably, said permeabilizing step comprises wiping the tissue section dry and circling the tissue section with an immunohistochemical pen, placing the tissue section flat in a wet box (with a small amount of water added), then applying a 1% Triton X-100 solution drop-wise to each circle of the tissue section of any value (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. Mu.l) of any value (e.g., 10, 11, 12, 13, 14, 15 min) in a wet box, permeabilizing the tissue section at room temperature for 5 to 15min (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min), placing the tissue section on a staining rack in a staining jar with TBST solution, and gently shaking the wash (preferably, said wash comprises 1 to 5 washes, further preferably 3 washes, 1 to 10, preferably 5min each wash);

6) And (3) sealing: dripping 10-30 μ l of blocking solution (such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 μ l) into the tissue section, incubating for 0.5-2h of blocking solution (such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 h), preferably, dripping the blocking solution into each circle of the tissue section;

7) Primary antibody incubation: dropping primary antibody of any value (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 μ l) into the tissue section after removing the blocking solution, and incubating at 4 ℃ for 4-24h (preferably 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24h, or incubating for 4h, and incubating overnight or incubating for 24 h), wherein the primary antibody incubating step further comprises placing the tissue section in a wet box and dropping the primary antibody into one circle of the tissue section;

8) And (3) secondary antibody incubation: after washing, dripping 10-30 μ l of secondary antibody with any value (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 μ l) into the tissue section, and incubating for 0.5-2h (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 h) in the absence of light, wherein preferably, the step of incubating the secondary antibody comprises washing in a TBST solution, and further preferably, the washing comprises washing for 1-5 times, more preferably 3 times, each time for 1-10min, preferably 5min each time;

9) Tabletting: after washing, dropwise adding a drop of anti-quenching agent into the tissue slice, then covering a cover glass to complete the slice preparation, wherein the slice preparation is finished in a dark environment, preferably, the slice preparation process comprises washing in a TBST solution, further preferably, the washing comprises washing for 1-5 times, more preferably, washing for 3 times, each time for 1-10min, preferably, each time for 5min, and preferably, the step of dropwise adding the primary anti-quenching agent is dropwise added into each circle of the tissue slice;

10 Microscopic examination photographed: immunofluorescent staining results of the tissue sections were photographed with a fluorescence microscope.

Preferably, the step 1) comprises:

1.1 Preparing a sealing liquid; preferably, the confining liquid comprises

5% -15% FBS, 25-35mg/mL BSA and PBS;

1.2 Preparing an antigen retrieval buffer; preferably, the antigen retrieval buffer comprises citric acid of any value (e.g., 0.32 to 0.40g/L, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40 g/L) and sodium citrate of any value (e.g., 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g/L) of 2.5 to 3.5g/L, and more preferably, the antigen retrieval buffer has a pH of 6.4;

1.3 Configure a primary antibody; comprising the steps of reacting a DAZL primary antibody: blocking solution =1: (20-200) preparing a solution, preferably 1: (25-100), for example 1: 20. 1: 30. 1: 40. 1:50. 1: 60. 1: 70. 1: 80. 1: 90. 1: 100. 1: 110. 1: 120. 1: 130. 1: 140. 1: 150. 1: 160. 1: 170. 1: 180. 1: 190. 1:200 of a carrier;

1.4 Preparing a secondary antibody; comprises the following steps of mixing a secondary antibody (preferably anti-mouse 488): blocking solution =1: (200-1000) configured as a solution, for example 1:200. 1: 300. 1: 400. 1: 500. 1: 600. 1: 700. 1: 800. 1: 900. 1:1000.

in a third aspect of the invention, a reagent for detecting a biomarker is provided.

Preferably, the agent detects the mRNA expression level of the DAZL gene and/or the SOX9 gene, and/or the expression level of the DAZL protein and/or the SOX9 protein.

Further preferably, the detection is the detection of the presence or absence of a biomarker.

Preferably, the reagent is used in immunofluorescence assay.

Preferably, the reagents used in the immunofluorescence assay include, but are not limited to, a blocking solution (prepared from 5% -15% FBS, 25-35mg/mL BSA and PBS), an antigen retrieval buffer (prepared from 0.32-0.40g/L citric acid, 2.5-3.5g/L sodium citrate, preferably, the pH of the antigen retrieval buffer is 6.4), a TBST solution, a 1% Triton X-100 solution, a primary antibody reagent and/or a secondary antibody reagent.

In a fourth aspect of the invention, there is provided a method of detecting germ cells, said method comprising detecting DAZL using immunofluorescence.

Preferably, the step of immunofluorescence is as described in the second aspect of the present invention.

Preferably, the germ cells can be germ cells derived from any tissue or organ, and in one embodiment of the invention, the germ cells are derived from testis or testis puncture extract.

In a fifth aspect of the invention, there is provided a method of detecting a support cell, said method comprising detecting SOX9 using immunofluorescence.

Preferably, the immunofluorescence procedure is as for the second aspect of the invention, but the primary antibody is an anti-SOX 9 antibody.

The process of configuring the primary antibody comprises the following steps of: blocking solution =1: (20-200) preparing a solution, preferably 1: (50-200), for example 1: 20. 1: 30. 1: 40. 1:50. 1: 60. 1: 70. 1: 80. 1: 90. 1: 100. 1: 110. 1: 120. 1: 130. 1: 140. 1: 150. 1: 160. 1: 170. 1: 180. 1: 190. 1:200 of a carrier;

preparing a secondary antibody; comprises the following steps of adding a secondary antibody (preferably anti-mouse 555): blocking solution =1: (200-1000) is configured as a solution, e.g. 1:200. 1: 300. 1: 400. 1: 500. 1: 600. 1: 700. 1: 800. 1: 900. 1:1000.

preferably, the support cell may be a support cell derived from any tissue or organ, and in one embodiment of the invention, the support cell is derived from testis or testis puncture extract.

In a sixth aspect of the invention, there is provided a diagnostic and/or prognostic article for azoospermia comprising a reagent for detecting DAZL and a reagent for detecting SOX9.

Preferably, the reagent for detecting DAZL and the reagent for detecting SOX9 are reagents according to the third aspect of the invention. Preferably, the product comprises a diagnostic kit, a gene chip, a microarray or a protein array.

In a seventh aspect of the invention, a method for the diagnostic and/or prognostic assessment of azoospermia is provided.

Preferably, the method comprises detecting the mRNA expression level of the DAZL gene and/or SOX9 gene, and/or the expression level of DAZL protein and/or SOX9 protein in the subject.

The term "diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression of a disease or likely progression in the future.

As used herein, "prognostic evaluation" refers to the evaluation of a patient's response to treatment, as well as the risk of future disease.

The terms "comprises" and "comprising" of this invention are open-ended descriptions that include the stated elements or steps, as well as other elements or steps that do not materially affect the properties of the invention.

All combinations of items described herein as "and/or" including the term connected should be considered as if each combination had been individually listed herein. For example, "A and/or B" includes "A", "A and B", and "B". As another example, "A, B and/or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

The individual can be a human or non-human animal, and the non-human animal can be a non-human mammal such as a mouse, a cow, a sheep, a rabbit, a pig, a monkey and the like.

The invention has the beneficial effects that:

by applying the technology of the invention, the semen-free patient can be assisted to make a decision without carrying out testicular incision surgery, and the pain and excessive medical behavior of the patient are reduced; the method helps doctors to judge the severity of azoospermia of patients, can guide the doctors to give the sterile patients appropriate hormone therapy, can also be used as a basis for further utilizing support cells to carry out alternative therapy in the future, and has very important clinical significance.

Meanwhile, the immunofluorescence staining method is improved, and the concentration of the reagent in the method is adjusted, so that the staining result is clearer.

Drawings

FIG. 1: HE staining results for sample 1;

FIG. 2 is a schematic diagram: HE staining results for sample 1;

FIG. 3: support cell immunofluorescence staining results for sample 1;

FIG. 4 is a schematic view of: germ cell immunofluorescence staining results for sample 1;

FIG. 5 is a schematic view of: HE staining results for sample 2;

FIG. 6: HE staining results for sample 2;

FIG. 7 is a schematic view of: the results of supported cellular immunofluorescence staining of sample 2;

FIG. 8: germ cell immunofluorescence staining results for sample 2;

FIG. 9: HE staining results for sample 3;

FIG. 10: HE staining results for sample 3;

FIG. 11: the results of supported cell immunofluorescence staining of sample 3;

FIG. 12: germ cell immunofluorescence staining results for sample 3;

FIG. 13 is a schematic view of: detecting the influence of overhigh antibody concentration in the dyeing process of the DAZL protein by an immunofluorescence method;

FIG. 14: immunofluorescence method for detecting influence of overhigh antibody concentration in dyeing process of SOX9 protein

FIG. 15: detecting the influence of too low antibody concentration in the dyeing process of the DAZL protein by an immunofluorescence method;

FIG. 16: and detecting the influence of too low antibody concentration in the dyeing process of the SOX9 protein by an immunofluorescence method.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1: application of fluorescent staining method for detecting DAZL protein and SOX9 protein in azoospermia detection

The sample in the invention is a testicle puncture sample of a non-obstructive azoospermia clinical patient, and the sample is embedded in paraffin and then dyed.

1. Preparation of reagents:

1.1 sealing liquid: 10% FBS 30mg/ml BSA + PBS

1.2 antigen retrieval buffer: 0.36g/L of citric acid; 3g/L sodium citrate, pH6.4.

1.3 preparation of primary antibody: DAZL (mouse anti-human): blocking liquid = 1; SOX9 (murine anti-human): blocking solution =1

1.4 preparation of secondary antibody: DAZL secondary antibody: anti-mouse 488: blocking solution = 1; SOX9 secondary antibody, anti-mouse 555: blocking solution = 1;

2. the operation steps are as follows:

2.1 baking sheet

And taking out the samples stored in a refrigerator at the temperature of-20 ℃, sequentially placing the samples on a dyeing rack, and drying the samples in an oven at the temperature of 65 ℃ for 2 hours.

2.2 dewaxing and rehydration

The staining rack is sequentially placed into a corresponding staining jar for paraffin section dewaxing and rehydration according to the sequence of the table 1:

TABLE 1

Reagent	Time
		Xylene 1	10min
Xylene 2	10min
		Anhydrous ethanol 1	5min
Absolute ethanol 2	5min
		90% ethanol	5min
80% ethanol	5min
		70% ethanol	5min
50% ethanol	5min
		Distilled water (three times)	5min

Note: in the step, the xylene and the alcohol with various concentrations can be recycled.

2.3 antigenic repair

500ml of antigen retrieval buffer solution is taken to be placed in an antigen retrieval box and is directly heated on an electromagnetic oven until boiling. And then placing the staining rack in an antigen retrieval box, covering and continuously heating for 10min, and taking out the antigen retrieval box for natural cold cutting. After cooling to room temperature, the staining rack was placed in a staining jar containing TBST solution and washed on a shaking table gently for 3 times, 5min each time.

2.4 permeation

After wiping the tissue sections dry, a circle is drawn around the tissue by an immunohistochemical pen, the tissue sections are placed in a wet box (a small amount of water is added), then 20 mu l of 1% Triton X-100 solution is dripped into each circle of the tissue sections, after the tissue sections are permeated for 10min at room temperature, the tissue sections are placed in a staining rack and then placed in a staining jar filled with TBST solution, and the tissue sections are gently shaken and washed for 3 times and 5min each time on a shaking table.

2.5 sealing

After being wiped dry, the tissue slices are horizontally placed in a wet box, 20 mu l of confining liquid is dripped into each circle of the tissue slices, and the tissue slices are incubated and sealed for 1h.

2.6 Primary antibody incubation

After gently wiping off the blocking solution, the tissue sections were placed flat in a wet box, 20. Mu.l of primary antibody solution (blocking solution preparation) was added dropwise to one circle of the tissue sections, 20. Mu.l of blocking solution was added dropwise to the other circle as a control, and the tissue sections were incubated overnight at 4 ℃.

2.7 incubation with Secondary antibody

The tissue slices were placed in a staining rack and then placed in a staining jar containing TBST solution and gently shaken and washed on a shaking table for 3 times, 5min each time. After wiping the tissue sections dry, the tissue sections are placed in a wet box, 20 mu l of secondary antibody solution (prepared by confining liquid) is dripped into each circle of the tissue sections, and the tissue sections are incubated for 1 hour in a dark place.

2.8 preparation of tablets

The tissue sections were placed on a staining rack and then placed in a staining jar containing TBST solution and gently shaken and washed on a shaker for 3 times, each time for 5min. The tissue slices are wiped dry and placed in a dry box, a drop of anti-quenching agent is dropped into each circle of the tissue slices, and then a cover glass is covered to complete the slide preparation.

Note: this step must be done in a dark environment.

2.9 microscopic examination and photograph

Immunofluorescent staining results of the tissue sections were photographed with a fluorescence microscope.

The results are shown in FIGS. 1-8, and FIGS. 1 and 2, FIGS. 5 and 6, FIGS. 9 and 10 are HE staining results for samples 1, 2 and 3, respectively, without spermatozoa nuclei, indicating that none of the three samples had sperm.

FIGS. 3 and 4 show the results of immunofluorescent staining of sample 1, and red fluorescent-supporting cells, i.e., SOX 9-positive, are seen in FIG. 3. A large number of green fluorescent germ cells, positive for DAZL, are visible in figure 4.

FIGS. 7 and 8 show the results of immunofluorescent staining of sample 2, and red fluorescent-supporting cells, i.e., SOX 9-positive, are shown in FIG. 7. In FIG. 8, germ cells without green fluorescence, i.e., negative to DAZL.

FIGS. 11 and 12 show the results of immunofluorescent staining of sample 3, and no red fluorescent support cells, i.e., SOX 9-negative, were found in FIG. 11. In FIG. 12, germ cells without green fluorescence, i.e., negative to DAZL.

Currently, the detection of azoospermia often requires the next detection step using invasive surgery (e.g., orchidrosis) after the azoospermia is confirmed by HE staining, and the methods described herein further classify patients after azoospermia is confirmed by HE staining on a molecular level.

In the present invention, definitive diagnosis and prognostic evaluation are performed by biomarker detection for non-obstructive azoospermia patients:

1. the first step is that the DAZL molecular marker detection shows that negative patients can be diagnosed as severe aspermia patients, and the diagnosis is carried out according to the molecular level without carrying out traumatic operations such as testicular dissection and the like;

2. for a patient with positive DAZL molecular marker detection, cells with reproductive function in tissues are indicated, although mature spermatids cannot be detected by HE staining, spermatogonial stem cells, spermatocytes and haploid spermatids may exist in the tissues of the patient, and the cells can develop into the mature spermatids under certain conditions, so that the SOX9 molecular marker is further detected to judge whether the tissues of the patient have spermatogenesis function, and if the SOX9 molecular marker is also positive, the tissues of the patient have the possibility of generating spermatids through the auxiliary function of the support cells.

Therefore, the invention can intuitively diagnose azoospermia and predict whether a patient can be treated by detecting the molecular level, thereby avoiding traumatic operation.

Control example 1 Effect of excessive antibody concentration on detection during immunofluorescence staining

The procedure for detecting the DAZL protein was compared to example 1 except that the concentration of the primary antibody was increased, and the other procedures were the same.

DAZL (mouse): blocking solution = 1;

SOX9 (mouse): blocking solution = 1.

As a result, as shown in FIGS. 13 and 14, the concentration of the primary antibody was too high, resulting in too strong background staining, and thus the stained cells could not be clearly judged. .

Comparative example 2 Effect of too low concentration of antibody on detection during immunofluorescence staining

The procedure for detecting the DAZL protein was as described in example 1, except that the concentration of the primary antibody was reduced, and the other procedures were the same.

DAZL (mouse) blocking solution = 1;

SOX9 (mouse): blocking solution = 1.

As a result, as shown in FIGS. 15 and 16, the primary antibody concentration was too low, and fluorescence staining was observed, but the staining intensity was too low to be recognized.

Although the present invention has been described in the foregoing by way of examples, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

1. Use of a biomarker or a reagent for detecting a biomarker, wherein the biomarker is DAZL, for the preparation of a product for the diagnostic and/or prognostic assessment of azoospermia.

2. The use of claim 1, wherein the biomarker further comprises SOX9.

3. The use of claim 2, wherein the reagent for detecting a biomarker is a reagent for detecting the mRNA expression level of the DAZL gene and/or the SOX9 gene, and/or the expression level of the DAZL protein and/or the SOX9 protein.

4. The use according to any one of claims 1 to 3, wherein the product comprises a diagnostic kit, a gene chip, a microarray or a protein array.

5. The use of claim 4, wherein the diagnostic kit comprises reagents for detecting DAZL using immunofluorescence.

6. The use according to any one of claims 4 to 5, wherein the diagnostic kit comprises reagents for detecting SOX9 using immunofluorescence.

7. The use of claim 5 or 6, wherein said immunofluorescence comprises:

1) Preparing a sealing liquid; the sealing liquid comprises

5% -15% FBS, 25-35mg/mL BSA and PBS;

2) Preparing an antigen retrieval buffer solution; the antigen retrieval buffer solution comprises 0.32-0.40g/L of citric acid and 2.5-3.5g/L of sodium citrate, and the pH value of the antigen retrieval buffer solution is 6.4;

3) Configuring a primary antibody; comprising the steps of reacting a DAZL primary antibody: confining liquid =1: (20-200) preparing into a solution;

4) Preparing a secondary antibody; comprises the following steps of: blocking solution =1: (200-1000) preparing into solution.

8. The detection method of claim 7, wherein the immunofluorescence assay further comprises:

1) Baking the slices: sequentially placing the samples on a dyeing rack, and drying the samples in a 65 ℃ oven for 2 hours;

2) Dewaxing and rehydration: putting the staining rack into a corresponding staining jar for dewaxing and rehydration of paraffin sections:

3) Antigen retrieval: placing the antigen repairing buffer solution into an antigen repairing box, heating to boil, placing the staining rack into the antigen repairing box, covering and continuously heating, taking out the antigen repairing box, naturally cold cutting, cooling to room temperature, placing the staining rack into a staining jar containing TBST solution, and slightly shaking and washing on a shaking table;

4) Permeability: dripping 1% Triton X-100 solution into the tissue slice, and permeating at room temperature;

5) And (3) sealing: dripping 10-30 μ l of confining liquid into the tissue slice, and incubating and confining for 0.5-2h;

6) Primary antibody incubation: after the confining liquid is removed, dripping 10-30 mu l of primary antibody into the tissue slice, and incubating for 4-24h at 4 ℃;

7) And (3) secondary antibody incubation: after washing, dripping 10-30 mul of secondary antibody into the tissue slice, and incubating for 0.5-2h in a dark place;

8) Tabletting: after washing, dripping a drop of anti-quenching agent into the tissue slice, and then covering a cover glass to finish the slice preparation which needs to be finished in a dark environment;

9) And (5) microscopic examination and photographing: immunofluorescent staining results of the tissue sections were photographed with a fluorescence microscope.

9. A product for the diagnostic and/or prognostic assessment of azoospermia, said product comprising reagents for the detection of DAZL and reagents for the detection of SOX9.

10. The product of claim 9, wherein the product comprises a diagnostic kit, a gene chip, a microarray or a protein array.

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