Tissue hexamine silver staining method

Abstract

The invention discloses a tissue hexamine silver staining method, and relates to the technical field of staining of animal tissue slices. The method comprises the following steps: taking a washed to-be-dyed tissue section, dropwise adding a periodate solution for treatment, soaking and washing; putting the tissue slice into a silver hexamine developing solution until the tissue slice is just immersed in the silver hexamine developing solution; heating and incubating; washing the developed tissue slice with water, and carrying out silver fixation treatment by using a sodium thiosulfate solution; and washing, dehydrating, dyeing, re-dehydrating, transparentizing with xylene, sealing and airing the tissue slice subjected to silver fixation treatment to finish dyeing. The invention provides a tissue hexamine silver staining method which solves the problem that a staining section is easy to dirty due to a silver mirror reaction in a traditional method. The developing time is short, the positive contrast after developing is strong, and the result is clear.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue section staining, and particularly to a tissue hexamine silver staining method. Background Art

[0002] Hexamine silver staining is also known as PASM (Periodic-acid silver methenamine) staining. It is mainly used for staining tissue basement membranes and fungi (mainly applied to Cryptococcus neoformans, Mucor, Aspergillus, Histoplasma, Penicillium marneffei, Actinomyces, etc.). The principle of hexamine silver staining is that periodic acid oxidizes with the tissue, exposing the aldehyde groups of the mucopolysaccharides in the tissue basement membrane or fungi. The aldehyde groups react with hexamine silver, reducing it to black metallic silver, and then using sodium thiosulfate to remove the unreacted silver ions.

[0003] Hexamine silver staining is a common staining method in tissue pathological section staining technology. It is used as a diagnostic basis for showing fungal and glomerular basement membrane changes in pathological diagnosis. However, due to the silver mirror reaction in the traditional hexamine silver staining method, there is a problem that the tissue sections are easily soiled in appearance and cannot be wiped off subsequently; and the color development time is relatively long, and the positive contrast degree after color development is not high. Summary of the Invention

[0004] Aiming at the above deficiencies of the prior art, the present invention provides a tissue hexamine silver staining method with a short color development time, strong positive contrast after color development, and clear results; specifically achieved through the following techniques.

[0005] A tissue hexamine silver staining method includes the following steps:

[0006] Take the washed tissue sections to be stained, drop and add periodic acid solution for treatment, and soak and wash.

[0007] Put the tissue sections into the hexamine silver color developing solution until the hexamine silver color developing solution just submerges the tissue sections; heat and incubate.

[0008] Wash the well-colored tissue sections with water, and perform silver fixation treatment with sodium thiosulfate solution.

[0009] Wash, dehydrate, stain the tissue sections after silver fixation treatment, dehydrate again, make them transparent with xylene, seal and fix, and air dry to complete the staining.

[0010] Furthermore, the concentration of the dropped periodic acid solution is 0.8 - 1.2%, and the treatment time is 12 - 16 min.

[0011] Furthermore, the concentration of the dropped periodic acid solution is 1.0%, and the treatment time is 14 min.

[0012] Further, the conditions for heating and incubation are constant temperature heating and incubation at 60-70°C.

[0013] Furthermore, the conditions for heating and incubation are constant temperature heating and incubation at 63°C.

[0014] Further, first observe the color development every 10-15 minutes until the background of the tissue section is yellowish brown, and then examine the color development under the microscope.

[0015] Furthermore, observe the color development every 10 minutes.

[0016] Further, when examining the color development under the microscope, if the color development is insufficient, take out the tissue section, wash it, and then add the hexamine silver staining solution again for color development until the color development requirements are met.

[0017] Furthermore, when adding the hexamine silver staining solution again for color development, observe the color development every 3-8 minutes.

[0018] Preferably, when adding the hexamine silver staining solution again for color development, observe the color development every 5 minutes.

[0019] Optionally, for the staining process after silver fixation, any one of the counterstaining methods such as eosin, light green, nuclear fast red, hematoxylin, etc. can be selected.

[0020] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides a method for hexamine silver staining of tissues, which solves the problem that the stained sections are easily soiled due to the silver mirror reaction in the traditional method; the color development time is short, the positive contrast after color development is strong, and the results are clear. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a 200-fold microscopic examination photo of a kidney tissue section;

[0022] Figure 2 It is a 400-fold microscopic examination photo of a kidney tissue section. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0024] In the following examples, all reagent raw materials used are purchased from Sinopharm Chemical Reagent Co., Ltd.

[0025] Example 1

[0026] The tissue hexamine silver staining method provided by this embodiment includes the following steps:

[0027] S1. Take a paraffin section of kidney tissue, dewax it, and wash it with tap water for 2 minutes.

[0028] S2. Immerse the section in pure water (C'estbon) and wash it 3 times, 1 minute each time.

[0029] Generally, the washing time can be 30 seconds to 1 minute.

[0030] S3. Use a group painting brush to draw a circle to enclose the tissue area to be stained, drop hyperiodic acid solution (concentration 1%), and oxidize it at room temperature for 14 minutes.

[0031] S4. Add it to pure water and soak and wash it 3 times, 1 minute each time.

[0032] S5. Take 20 ml of hexamine silver chromogenic solution (Shanghai Shangbao Company), pour it into the section box containing the section until the tissue is just submerged; incubate it in a water bath at 63°C.

[0033] At the beginning, observe the chromogenic situation every 10 minutes until the background of the section is yellowish brown, then examine the chromogenic situation under the microscope. If the chromogenic is not enough, wash it clean with pure water and then put it into the chromogenic solution to continue chromogenic, observe the chromogenic situation every 5 minutes until the glomerular and tubular basement membranes are black, the glomerular capsule membrane is black, the microvessels are black, and the background is golden yellow to brownish yellow.

[0034] S6. Wash the well-chromed tissue section clean with pure water, treat it with hypo solution (sodium thiosulfate solution) for 1 minute until the background is preferably light yellowish brown or colorless, the positive is black, and the contrast is clear.

[0035] Generally, the treatment time with hypo solution (sodium thiosulfate solution) can be 30 seconds to 1 minute.

[0036] S7. Wash the section clean with pure water, dehydrate it in two cylinders of absolute ethanol, then stain it with eosin stain for about 30 seconds, dehydrate it in the third cylinder of absolute ethanol, and clear it with xylene; finally, seal it wet with neutral gum and let it dry to complete the staining of the tissue section.

[0037] In addition to eosin stain, any one of the counterstaining methods such as light green, nuclear fast red, hematoxylin, etc. can also be selected for the staining method used.

[0038] The staining result is as Figure 1 、 2 shown. In the kidney tissue section, the glomerulus (indicated by the red arrow), the basement membranes of the renal tubules (indicated by the yellow arrow) are black, the glomerular capsule membrane is black, the microvessels are black (indicated by the black arrow); if there are fungi, the fungi are black rod-shaped (indicated by the green arrow), connected head to tail; the background is red.

[0039] When the lung tissue sections are stained by the above method, it can be found that the fungal hyphae are black rod-shaped, the fungal spores are black, and the background is red.

[0040] Example 2

[0041] The difference between this example and Example 1 is that: the concentration of the periodic acid solution used is 1.2%, the treatment time is 12 min; the heating incubation temperature is kept constant at 70 °C.

[0042] Example 3

[0043] The difference between this example and Example 1 is that: the concentration of the periodic acid solution used is 0.8%, the treatment time is 16 min; the heating incubation temperature is kept constant at 60 °C.

[0044] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. A tissue hexamine silver staining method, characterized in that, Comprising the following steps: Take the washed tissue section to be stained, drop and add periodic acid solution for treatment, soak and wash; Put the tissue section into the hexamine silver chromogenic solution until the hexamine silver chromogenic solution just submerges the tissue section; Heat and incubate; Wash the well-chromed tissue section with water and perform silver fixation treatment with sodium thiosulfate solution; Wash, dehydrate, stain the tissue section after silver fixation treatment, dehydrate again, make it transparent with xylene, seal, and air dry to complete the staining.

2. The tissue hexamine silver staining method according to claim 1, wherein The concentration of the dropped periodic acid solution is 0.8 - 1.2%, and the treatment time is 12 - 16 min.

3. The periodic acid-silver methenamine staining method for tissues according to claim 1, characterized in that, The concentration of the dropped periodic acid solution is 1.0%, and the treatment time is 14 min.

4. The tissue hexamine silver staining method according to claim 1, characterized in that, The conditions for the heat incubation are constant temperature heating incubation at 60 - 70 °C.

5. The tissue hexamine silver staining method according to claim 4, wherein The conditions for the heat incubation are constant temperature heating incubation at 63 °C.

6. The method for tissue hexamine silver staining according to claim 1, characterized in that, First, observe the chromogenic situation every 10 - 15 min, and microscopically examine the chromogenic situation when the background of the tissue section is yellowish brown.

7. The tissue hexamine silver staining method according to claim 6, wherein Observe the chromogenic situation every 10 min.

8. The tissue hexamine silver staining method according to claim 6 or 7, characterized in that, When microscopically examining the chromogenic situation, if the chromogenesis is insufficient, take out the tissue section, wash it, and then add the hexamine silver chromogenic solution again for chromogenesis until the chromogenic requirements are met.

9. The tissue hexamine silver staining method according to claim 8, wherein, When adding the hexamine silver chromogenic solution again for chromogenesis, observe the chromogenic situation every 3 - 8 min.

10. The tissue hexamine silver staining method according to claim 9, characterized in that, When adding the hexamine silver chromogenic solution again for chromogenesis, observe the chromogenic situation every 5 min.

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