A method for using a frozen section in gori's staining

Abstract

The application provides a method for applying frozen sections in Golgi staining, which comprises the following steps: sample taking, immersion, soaking in a cryoprotective solution, frozen sectioning, staining, dehydration and mounting. The application is simple, practical and easy to operate.

Description

Technical Field

[0001] This invention belongs to the field of Golgi staining technology, specifically relating to a method for using frozen sections in Golgi staining. Background Technology

[0002] Existing methods for frozen sectioning in Golgi staining involve complex processes of embedding tissue sections in double-distilled water and mounting them, leading to difficulties in reproducibility and requiring strict adherence to procedures. Otherwise, severe detachment occurs later, and the resulting dehydration and clearing can cause tissue shrinkage, resulting in poor slide preparation. Currently, there is no solution to the problem of detachment. Some commercial kits offer brief instructions for frozen sectioning, but they are expensive and not widely adopted. Paraffin sectioning and vibratory sectioning methods are also available, but paraffin sections cannot achieve a thickness of more than 60 μm, hindering dendritic branching and axonal analysis. Vibratory microtome machines are also expensive, limiting their widespread adoption. Therefore, it remains necessary to innovate a simpler, more practical, and easier-to-use Golgi-Cox staining protocol. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for using frozen sections in Golgi staining, which is simple, practical and easier to operate, in order to address the shortcomings of the prior art.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for using frozen sections in Golgi staining, the method being as follows:

[0005] S1. Material collection: Brain tissue from C57BL / 6J mice was collected, washed with double-distilled water, and the brain area was cut into blocks with a thickness of 0.5cm.

[0006] S2, Immersion: The brain tissue block obtained in S1 is placed in a centrifuge tube containing working solution and soaked for 6 to 24 hours. The working solution is then replaced. The working solution is not replaced thereafter. The soaking time is 7 to 14 days. The brain tissue block is stored at room temperature away from light to obtain the soaked brain tissue block.

[0007] The working solution is prepared by mixing solution A, solution B, solution C and double-distilled water in a volume ratio of 5:5:4:10 by shaking, reacting at room temperature in the dark for 24-48 hours, and then filtering with a 0.22μmm filter or filter paper to obtain the working solution.

[0008] Solution A is prepared by dissolving 5g of potassium dichromate in 100mL of double-distilled water;

[0009] Solution B is prepared by dissolving 5g of mercuric chloride in 100mL of double-distilled water under water bath heating at 90℃.

[0010] Solution C is prepared by dissolving 5g of potassium chelate in 100mL of double-distilled water;

[0011] S3. Immerse in cryoprotectant solution:

[0012] Under 100 Lux of light, the brain tissue block after soaking in S2 was removed with plastic clamps, dried with absorbent paper, and then soaked in cryoprotectant for 2 to 3 days. The cryoprotectant was replaced every 24 hours until the brain tissue block sank to the bottom, thus obtaining the brain tissue block soaked in cryoprotectant.

[0013] The cryoprotectant is made from the following raw materials in the following amounts: 20g sucrose, 15mL-20mL glycerin, and diluted to 100mL with double-distilled water.

[0014] S4, Frozen slices:

[0015] S401. In a cryostat at a temperature of -25℃ to -20℃, OCT embedding agent is dropped onto a pre-cooled tray to form an ice bottom. Then, the brain tissue block obtained in S3 after soaking in cryoprotectant is fixed on the tray. After dropping OCT embedding agent onto the brain tissue block after soaking in cryoprotectant, it is frozen in the cryostat for more than 30 minutes to obtain a frozen brain tissue block.

[0016] S402. The frozen brain tissue block from S401 is sliced ​​using the cryostat. After slicing, the edges are held by tweezers and the slices are placed in a well plate containing 30% sucrose solution to obtain the slices.

[0017] The 30% sucrose solution was prepared by dissolving 30g of sucrose in 100mL of double-distilled water.

[0018] S403. Using a Pasteur pipette, draw 30% sucrose solution onto a glass slide coated with gelatin to form a water area. Using a frozen sectioning blade, move the section obtained in S402 onto the glass slide, flatten the section, discard the excess sucrose solution, and then adjust the position of the section with a needle or transfer the tissue into a dish containing 30% sucrose solution to retrieve the section so that it adheres to the glass slide coated with gelatin, thus obtaining the mounted section.

[0019] S404. Place the pasted film obtained in S403 in a dark place, under room temperature and ventilation conditions, overnight, or bake at 37°C for 30 to 60 minutes to obtain the dried film.

[0020] S5, Staining:

[0021] S501. Wash the dried tablets obtained in S404 twice with double-distilled water, 5 minutes each time;

[0022] S502. After staining in a staining jar containing ammonia solution in the dark for 10 minutes, wash twice with double-distilled water for 5 minutes each time.

[0023] The ammonia solution is prepared by mixing ammonia water and double-distilled water in a volume ratio of 3:1 with a mass fraction of 25% to 28%.

[0024] S503. Wash the tablets in 1% sodium thiosulfate solution for at least 15 minutes.

[0025] S504. After washing, place the slides on a rack, place the rack horizontally in double-distilled water with the tissue side facing up, and wash twice, 5 minutes each time.

[0026] S505. Place the film in an oven at 60℃ and bake in the dark for 2 to 3 hours.

[0027] S506, wash with water 4 times, 5 minutes each time;

[0028] S6. Dehydration and Installation:

[0029] S601, Dehydration and Clearing: Place the stained slides obtained in S506 in the following solutions in sequence for dehydration and clearing: 50% ethanol aqueous solution for 5 min, 75% ethanol aqueous solution for 5 min, 95% ethanol aqueous solution for 5 min, anhydrous ethanol A1 for 5 min, anhydrous ethanol A2 for 5 min, xylene I for 5 min, and xylene II for 5 min.

[0030] S602. Mount the slide with neutral resin and store in the dark until microscopic examination.

[0031] Preferably, the perforated plate in S402 is a 6-well plate or a 12-well plate.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] This invention uses an OCT embedding method, reducing the difficulty of ice embedding. Many laboratories still face limitations such as the limited time available for cryostats; the introduction of the slide-retrieval method solves this problem. Our experiments have shown that using 12-well or 6-well plates allows for both good slide development and easy transport. Existing slide mounting methods are difficult, involve multiple steps, and are complex, while the mounting method in this invention is simpler and easier to learn. Furthermore, methods for preventing slide detachment and post-detachment treatment are provided. The staining method in this invention shortens the staged washing time and incorporates a post-staining baking step, effectively solving the problem of subsequent slide detachment without affecting the staining effect.

[0034] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0035] Figure 1 This is the unfolding process of Embodiment 1 of the present invention.

[0036] Figure 2 This describes the patching process and drying standards of Embodiment 1 of the present invention.

[0037] Figure 3 This describes the tissue staining change process in Example 1 of the present invention.

[0038] Figure 4 This is the Golgi staining result of Example 1 of the present invention. Detailed Implementation

[0039] Example 1

[0040] The method for using frozen sections in Golgi staining in this embodiment is as follows:

[0041] S1. Material collection: Brain tissue from C57BL / 6J mice was collected, washed with double-distilled water, and the brain area was cut into blocks with a thickness of 0.5cm.

[0042] S2, Immersion: The brain tissue block obtained in S1 was placed in a centrifuge tube containing working solution and soaked for 24 hours. The working solution was then replaced, and the working solution was not replaced thereafter. The soaking was carried out for a total of 14 days. The brain tissue block was stored at room temperature and protected from light to obtain the brain tissue block after immersion.

[0043] The working solution is prepared by mixing solution A, solution B, solution C and double-distilled water in a volume ratio of 5:5:4:10 by shaking, reacting at room temperature in the dark for 24 hours, and then filtering with a 0.22μmm filter to obtain the working solution.

[0044] Solution A is prepared by dissolving 5g of potassium dichromate in 100mL of double-distilled water;

[0045] Solution B is prepared by dissolving 5g of mercuric chloride in 100mL of double-distilled water under water bath heating at 90℃; S3, Immersion in cryoprotectant:

[0046] Under 100 Lux illumination, the brain tissue block obtained from S2 after soaking in the working solution was removed with plastic clamps. After drying with absorbent paper, the dried brain tissue block was placed in the cryoprotectant for 2 days. The process should be gentle (brain tissue soaked in the working solution is particularly fragile). The cryoprotectant was changed every 24 hours until the brain tissue block sank to the bottom, at which point it could be sectioned to obtain the brain tissue block soaked in the cryoprotectant.

[0047] The cryoprotectant is made from the following raw materials in the following amounts: 20g sucrose, 15mL glycerol, and diluted to 100mL with double-distilled water.

[0048] S4, Frozen slices:

[0049] S401. In a cryostat at -20°C, OCT embedding agent is dropped onto a pre-cooled tray to form an ice bottom. Then, the brain tissue block obtained in S3 after soaking in cryoprotectant is fixed on the tray. After dropping OCT embedding agent onto the brain tissue block soaked in cryoprotectant, an adhesion layer of about 2 mm is formed around the brain tissue. The tissue block is frozen in the cryostat for 30 minutes to obtain a frozen brain tissue block.

[0050] This embodiment uses the OCT embedding method, which reduces the difficulty of ice embedding. Ice embedding is rare in frozen sections. After embedding in disposable embedding dishes, trimming may cause the ice to break, which will affect the sectioning. Moreover, it is difficult to control the force when sectioning. Cutting may cause the ice in some areas to break and the surrounding tissue to be exposed. Excessive exposure may cause the tissue to fall off. It takes a certain amount of time to master the sectioning technique, and it is quite difficult to get started.

[0051] S402. Slice the frozen brain tissue block from S401 using the cryostat, as shown in the example. Figure 1 As shown in A, after slicing, the edges are held by tweezers and the slice is placed into a 6-well plate containing 30% sucrose solution for spreading. The wells are as follows: Figure 1 As shown in B, the tissue can be gently moved with tweezers to help spread the tissue and obtain sections; as... Figure 1 As shown in C; when sectioning, if the tissue is too sticky to spread, the freezing time should be extended; if rigid sections are produced, the immersion time in the cryoprotectant should be extended and the cryoprotectant should be tested for quality. Figure 1 The text describes the unfolding process, where A represents the tissue obtained after being cut by a cryostat; B represents a well plate containing an appropriate volume of sucrose; and C represents the unfolded tissue, which should be smooth, flat, and intact.

[0052] The 30% sucrose solution was prepared by dissolving 30g of sucrose in 100mL of double-distilled water.

[0053] S403. Using a Pasteur pipette, draw 30% sucrose solution onto a glass slide coated with gelatin to form a watery area. Figure 2 As shown in C, use a frozen sectioning blade to transfer the section obtained in S402 onto a glass slide, flatten the section, and discard any excess sucrose solution. Figure 2 B, C, D, and E. Then adjust the position of the sections using a needle. Alternatively, the tissue can be transferred to a dish containing 30% sucrose solution and the sections can be retrieved so that they adhere to a glass slide coated with gelatin, resulting in a properly mounted slide. Figure 2 As shown in A and E;

[0054] This embodiment uses the slide-catching method, which ensures that slides do not need to be immediately mounted during slicing, making them convenient to carry and store. This allows the cryostat to be used for slicing without wasting time on mounting slides, thus solving the problem of limited usage time for cryostats.

[0055] S404. Place the pasted film obtained in S403 in a dark place, under room temperature and ventilation conditions, overnight to obtain a dried film; Figure 2 As shown in Figure F, the standard is that the tissue on the slide shows signs of drying. Figure 2 As indicated by the two arrows on the left in :G, you can stop baking now. Baking for too long will cause the texture to dry and crack, compromising its integrity. Figure 2 The two arrows on the right side of :G indicate this;

[0056] In this step, the pasted film can also be placed in a dark place and baked at 37℃ for 30 to 60 minutes to obtain a dried film;

[0057] Figure 2 This section describes the tissue mounting process and drying standards; where A: represents retrieving the tissue from the dish; B: represents retrieving the tissue from the well plate; C: represents the process of transferring the tissue with a blade into the water on a glass slide coated with gelatin; D: represents flattening the tissue on the glass slide; E: represents an image of the mounted tissue; F: represents the dried tissue; G: represents a comparison of the degree of tissue drying.

[0058] S5, Staining:

[0059] S501. Wash the dried tablets obtained in S404 twice with double-distilled water, 5 minutes each time;

[0060] S502. Stain in a staining vat containing ammonia solution. The stained tissue is as follows: Figure 3 As shown in A, after staining in the dark for 10 minutes, wash twice with double-distilled water for 5 minutes each time;

[0061] The ammonia solution is prepared from ammonia water with a mass fraction of 25% and double-distilled water in a volume ratio of 3:1;

[0062] Ammonia solutions are prepared and used under the protection of fume hoods and protective gloves.

[0063] S503. Wash the tablets in 1% sodium thiosulfate solution for at least 15 minutes.

[0064] The method for preparing a 1% sodium thiosulfate solution is to dissolve 3.14 g of sodium thiosulfate pentahydrate in 200 mL of double-distilled water, and to prepare and use the solution under the protection of a fume hood and protective gloves.

[0065] At this point, it's important to note that the washed tissue should be predominantly white. The more yellow present, the worse the washing process. This is because tissue containing yellow material will crack after baking and form impurities after sealing, negatively impacting the slide quality. Figure 3 As shown in Figure B, the two arrows on the left indicate tissues that failed the washing test, while the two arrows on the right indicate tissues that passed the washing test.

[0066] S504. After washing, place the slides on a rack horizontally in double-distilled water, with the tissue side facing up. Wash twice, 5 minutes each time. If the slides fall off, place them in the oven to bake. At this point, the slides are very prone to falling off during washing, but remounting is allowed because the tissue is less rigid and more flexible than when sectioned, making it less susceptible to damage. If the tissue is uneven when mounted, you can remount it after it falls off. Microscopic examination can then be performed; the morphology of neurons can now be clearly seen. Figure 3 As shown in C;

[0067] S505. Place the slices in an oven at 60℃ and bake in the dark for 2 hours, until completely dehydrated. This effectively prevents the slices from falling off. The texture after baking is as follows: Figure 3 As shown in D, the tissue should be free of cracks at this point. If cracks are present, refer to S503 to compare whether the film processing is up to standard.

[0068] Figure 3 The images show the process of tissue staining changes; A: a tissue image stained with ammonia solution; B: a comparison image of tissue state after washing with sodium thiosulfate pentahydrate; C: a microscopic image after washing with water; D: a tissue image after baking.

[0069] S506, wash with water 4 times, 5 minutes each time;

[0070] This step can also be optional, depending on your needs, by using 1% cresol purple staining.

[0071] S6. Dehydration and Installation:

[0072] S601, Dehydration and Clearing: Place the stained slides obtained in S506 in the following solutions in sequence for dehydration and clearing: 50% ethanol aqueous solution for 5 min, 75% ethanol aqueous solution for 5 min, 95% ethanol aqueous solution for 5 min, anhydrous ethanol A1 for 5 min, anhydrous ethanol A2 for 5 min, xylene I for 5 min, and xylene II for 5 min.

[0073] S602. Mount the slide with neutral resin and store in the dark until microscopic examination.

[0074] Figure 4The images shown are Golgi staining results from this embodiment, where A: shows an image of cortical neurons; B: shows an image of hippocampal neurons; C: shows an image of hypothalamic neurons; D: shows an image of Purkinje neurons; and E and F: show images of dendritic spines.

[0075] Staining results as follows Figure 4 As shown, we used laser confocal microscopy to observe clearly stained cortical neurons (A), hippocampal neurons (B), and hypothalamic neurons (C) at 50 μm; cerebellar Purkinje neurons (D) at 20 μm; and clearly stained dendritic spines at 20 μm (E) and 2 μm (F). The above images were taken after the prepared slides were stored at room temperature in the dark for two months, demonstrating that the stained slides prepared in this embodiment have a long storage time and produce few impurities, which do not affect the analysis of dendritic spines.

[0076] Example 2

[0077] The method for using frozen sections in Golgi staining in this embodiment is as follows:

[0078] S1. Material collection: Brain tissue from C57BL / 6J mice was collected, washed with double-distilled water, and the brain area was cut into blocks with a thickness of 0.5cm.

[0079] S2, Immersion: The brain tissue block obtained in S1 was placed in a centrifuge tube containing working solution and soaked for 6 hours. The working solution was then replaced, and the working solution was not replaced thereafter. The soaking was carried out for a total of 7 days. The brain tissue block was stored at room temperature and protected from light to obtain the brain tissue block after immersion.

[0080] The working solution is prepared by mixing solution A, solution B, solution C and double-distilled water in a volume ratio of 5:5:4:10 by shaking, reacting at room temperature in the dark for 48 hours, and then filtering with filter paper to obtain the working solution.

[0081] Solution A is prepared by dissolving 5g of potassium dichromate in 100mL of double-distilled water;

[0082] Solution B is prepared by dissolving 5g of mercuric chloride in 100mL of double-distilled water under water bath heating at 90℃.

[0083] Solution C is prepared by dissolving 5g of potassium chelate in 100mL of double-distilled water;

[0084] S3. Immerse in cryoprotectant solution:

[0085] Under low light conditions of 100 Lux, the brain tissue block after soaking in S2 was removed with plastic clamps, dried with absorbent paper, and then immersed in cryoprotectant for 3 days. The cryoprotectant was replaced every 24 hours until the brain tissue block sank to the bottom, thus obtaining the brain tissue block after soaking in cryoprotectant.

[0086] The cryoprotectant is made from the following raw materials in the following amounts: 20g sucrose, 20mL glycerin, and diluted to 100mL with double-distilled water;

[0087] S4, Frozen slices:

[0088] S401. In a cryostat at -25°C, OCT embedding agent is dropped onto a pre-cooled tray to form an ice bottom. Then, the brain tissue block soaked in cryoprotectant obtained in S3 is fixed on the tray. After dropping OCT embedding agent onto the brain tissue block soaked in cryoprotectant, it is frozen in the cryostat for 40 minutes to obtain the frozen brain tissue block.

[0089] S402. Slice the frozen brain tissue block from S401 using the cryostat, as shown in the example. Figure 1 As shown in A, after slicing, the edges are held by tweezers and the slices are placed into a 12-well plate containing 30% sucrose solution to obtain the slices.

[0090] The 30% sucrose solution was prepared by dissolving 30g of sucrose in 100mL of double-distilled water.

[0091] S403. Using a Pasteur pipette, draw 30% sucrose solution onto a glass slide coated with gelatin to form a water area. Using a frozen sectioning blade, move the section obtained in S402 onto the glass slide, flatten the section, discard the excess sucrose solution, and then adjust the position of the section with a needle or transfer the tissue into a dish containing 30% sucrose solution to retrieve the section so that it adheres to the glass slide coated with gelatin, thus obtaining the mounted section.

[0092] S404. Place the pasted film obtained in S403 in a dark place and bake at 37℃ for 60 minutes to obtain the dried film.

[0093] S5, Staining:

[0094] S501. Wash the dried tablets obtained in S404 twice with double-distilled water, 5 minutes each time;

[0095] S502. Stain in a staining vat containing ammonia solution. The stained tissue is as follows: Figure 3 As shown in A, after staining in the dark for 10 minutes, wash twice with double-distilled water for 5 minutes each time;

[0096] The ammonia solution is prepared from ammonia water with a mass fraction of 28% and double-distilled water in a volume ratio of 3:1;

[0097] S503. Wash the tablets in 1% sodium thiosulfate solution for at least 15 minutes.

[0098] S504. After washing, place the slides on a rack, place the rack horizontally in double-distilled water with the tissue side facing up, and wash twice, 5 minutes each time.

[0099] S505. Place the film in an oven at 60℃ and bake in the dark for 3 hours.

[0100] S506, wash with water 4 times, 5 minutes each time;

[0101] S6. Dehydration and Installation:

[0102] S601, Dehydration and Clearing: Place the stained slides obtained in S506 in the following solutions in sequence for dehydration and clearing: 50% ethanol aqueous solution for 5 min, 75% ethanol aqueous solution for 5 min, 95% ethanol aqueous solution for 5 min, anhydrous ethanol A1 for 5 min, anhydrous ethanol A2 for 5 min, xylene I for 5 min, and xylene II for 5 min.

[0103] S602. Mount the slide with neutral resin and store in the dark until microscopic examination.

[0104] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for using frozen sections in Golgi staining, characterized in that, The method is as follows: S1. Material collection: Brain tissue from C57BL / 6J mice was collected, washed with double-distilled water, and the brain area was cut into blocks with a thickness of 0.5cm. S2, Immersion: The brain tissue block obtained in S1 is placed in a centrifuge tube containing working solution and soaked for 6 to 24 hours. The working solution is then replaced. The working solution is not replaced thereafter. The soaking time is 7 to 14 days. The brain tissue block is stored at room temperature away from light to obtain the soaked brain tissue block. The working solution is prepared by mixing solution A, solution B, solution C and double-distilled water in a volume ratio of 5:5:4:10 by shaking, reacting at room temperature in the dark for 24-48 hours, and then filtering with a 0.22μmm filter or filter paper to obtain the working solution. Solution A is prepared by dissolving 5g of potassium dichromate in 100mL of double-distilled water; Solution B is prepared by dissolving 5g of mercuric chloride in 100mL of double-distilled water under water bath heating at 90℃. Solution C is prepared by dissolving 5g of potassium chromate in 100mL of double-distilled water; S3. Immerse in cryoprotectant solution: Under 100 Lux of light, the brain tissue block after soaking in S2 was removed with plastic clamps, dried with absorbent paper, and then soaked in cryoprotectant for 2 to 3 days. The cryoprotectant was replaced every 24 hours until the brain tissue block sank to the bottom, thus obtaining the brain tissue block soaked in cryoprotectant. The cryoprotectant is made from the following raw materials in the following amounts: 20g sucrose, 15mL~20mL glycerin, and diluted to 100mL with double-distilled water. S4, Frozen slices: S401. In a cryostat at a temperature of -25℃ to -20℃, OCT embedding agent is dropped onto a pre-cooled tray to form an ice bottom. Then, the brain tissue block obtained in S3 after soaking in cryoprotectant is fixed on the tray. After dropping OCT embedding agent onto the brain tissue block soaked in cryoprotectant, it is frozen in the cryostat for more than 30 minutes to obtain a frozen brain tissue block. S402. The frozen brain tissue block from S401 is sliced ​​using the cryostat. After slicing, the edges are held by tweezers and the slices are placed in a well plate containing 30% sucrose solution to obtain the slices. The 30% sucrose solution is prepared by dissolving 30g of sucrose in 100mL of double-distilled water; S403. Using a Pasteur pipette, draw 30% sucrose solution onto a glass slide coated with gelatin to form a water area. Using a frozen sectioning blade, move the section obtained in S402 onto the glass slide, flatten the section, discard the excess sucrose solution, and then adjust the position of the section with a needle or transfer the tissue into a dish containing 30% sucrose solution to retrieve the section so that it adheres to the glass slide coated with gelatin, thus obtaining the mounted section. S404. Place the pasted film obtained in S403 in a dark place, under room temperature and ventilation conditions, overnight, or bake at 37℃ for 30 to 60 minutes to obtain the dried film. S5, Staining: S501. Wash the dried tablets obtained in S404 twice with double-distilled water, 5 minutes each time; S502. After staining in a staining jar containing ammonia solution in the dark for 10 minutes, wash twice with double-distilled water for 5 minutes each time. The ammonia solution is prepared by mixing ammonia water and double-distilled water at a volume ratio of 3:1 with a mass fraction of 25% to 28%. S503. Wash the tablets in 1% sodium thiosulfate solution for at least 15 minutes. S504. After washing, place the slides on a rack, place the rack horizontally in double-distilled water with the tissue side facing up, and wash twice, 5 minutes each time. S505. Place the film in an oven at 60℃ and bake in the dark for 2-3 hours. S506, wash with water 4 times, 5 minutes each time; S6. Dehydration and Installation: S601, Dehydration and Clearing: Place the stained slides obtained in S506 in the following solutions in sequence for dehydration and clearing: 50% ethanol aqueous solution for 5 min, 75% ethanol aqueous solution for 5 min, 95% ethanol aqueous solution for 5 min, anhydrous ethanol A1 for 5 min, anhydrous ethanol A2 for 5 min, xylene I for 5 min, and xylene II for 5 min; S602. Mount the slide with neutral resin and store in the dark until microscopic examination.

2. The method for using frozen sections in Golgi staining according to claim 1, characterized in that, The perforated plate mentioned in S402 is a 6-well plate or a 12-well plate.

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