CN118090377A - Kit and method for special staining of kidney puncture tissue biopsy lens - Google Patents
Kit and method for special staining of kidney puncture tissue biopsy lens Download PDFInfo
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- CN118090377A CN118090377A CN202410516514.6A CN202410516514A CN118090377A CN 118090377 A CN118090377 A CN 118090377A CN 202410516514 A CN202410516514 A CN 202410516514A CN 118090377 A CN118090377 A CN 118090377A
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- 238000010186 staining Methods 0.000 title claims abstract description 117
- 210000003734 kidney Anatomy 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 45
- 238000001574 biopsy Methods 0.000 title claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 102
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 65
- 238000004043 dyeing Methods 0.000 claims abstract description 56
- 239000008367 deionised water Substances 0.000 claims abstract description 54
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 54
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 claims abstract description 43
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 38
- 229960000583 acetic acid Drugs 0.000 claims abstract description 19
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 19
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 claims abstract description 12
- RZUBARUFLYGOGC-MTHOTQAESA-L acid fuchsin Chemical compound [Na+].[Na+].[O-]S(=O)(=O)C1=C(N)C(C)=CC(C(=C\2C=C(C(=[NH2+])C=C/2)S([O-])(=O)=O)\C=2C=C(C(N)=CC=2)S([O-])(=O)=O)=C1 RZUBARUFLYGOGC-MTHOTQAESA-L 0.000 claims abstract description 10
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000007447 staining method Methods 0.000 claims abstract description 9
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 claims abstract description 9
- KHOITXIGCFIULA-UHFFFAOYSA-N Alophen Chemical compound C1=CC(OC(=O)C)=CC=C1C(C=1N=CC=CC=1)C1=CC=C(OC(C)=O)C=C1 KHOITXIGCFIULA-UHFFFAOYSA-N 0.000 claims abstract description 3
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- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 50
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 claims description 46
- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical compound [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 claims description 44
- 229910052808 lithium carbonate Inorganic materials 0.000 claims description 44
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 claims description 36
- 239000012188 paraffin wax Substances 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
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- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 24
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 24
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- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims description 16
- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 16
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 claims description 15
- IRERQBUNZFJFGC-UHFFFAOYSA-L azure blue Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[S-]S[S-].[O-][Si]([O-])([O-])[O-].[O-][Si]([O-])([O-])[O-].[O-][Si]([O-])([O-])[O-].[O-][Si]([O-])([O-])[O-].[O-][Si]([O-])([O-])[O-].[O-][Si]([O-])([O-])[O-] IRERQBUNZFJFGC-UHFFFAOYSA-L 0.000 claims description 14
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- 239000004312 hexamethylene tetramine Substances 0.000 claims description 11
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 10
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 claims description 8
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a kit and a staining method for special staining of a kidney puncture tissue biopsy lens, belongs to the technical field of biological detection, and aims to solve the problems of insufficient staining of target substances and excessive staining of non-target substances in the existing staining kit. The kit comprises PASM sets of MASSON stain, MASSON stain and Congo red stain; the PASM sets of MASSON stain and MASSON stain comprise a first self-made reagent, a second self-made reagent and a third self-made reagent; the first homemade reagent comprises bisacodyl red, acid fuchsin, deionized water and glacial acetic acid; the second self-made reagent comprises phosphomolybdic acid, phosphotungstic acid and deionized water; the third self-made reagent comprises water-soluble aniline blue, deionized water and glacial acetic acid. The invention can realize proper coloring of target substances and does not excessively color non-target substances by the accurate design and dyeing method of the selection and the proportion of the constituent materials.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for special staining of a kidney puncture tissue biopsy lens and a staining method.
Background
The kidney puncture biopsy pathological examination mainly comprises routine HE staining, PASM sets of MASSN staining, PAS staining, MASSN staining, congo Red staining (Congo Red staining), immunohistochemical staining or immunofluorescence staining and electron microscopy, and the above examination complements each other, and is an essential examination for pathological diagnosis of kidney diseases. Wherein PASM sets of MASSON staining, PAS staining, MASSON staining and Congo Red staining belong to special staining, can intuitively display the pathological changes of glomerular basement membrane, injury of renal tubules, renal interstitial inflammation and fibrosis, the hardening degree of blood vessels, the deposition amount and the deposition position of special proteins and the like, and has very important significance for determining the pathological types of kidney diseases.
The existing special staining method of the kidney puncture tissue biopsy lens is mainly realized by self-made special staining reagent or existing commercial reagent kit, but PASM sets of MASSON staining reagent kit, MASSON staining reagent kit and Congo Red staining reagent kit which are available on the market at present have the problems of insufficient coloring of target substances and excessive coloring of non-target substances, can not clearly delineate pathological change positions, are widely used for all tissues, have no specific outlining on the structure of kidney tissues, seriously influence pathological diagnosis of doctors, influence formulation of treatment schemes and further influence treatment effects of patients. Thus, there is a need for optimizing specific stains for kidney-penetrating tissue biopsies.
Disclosure of Invention
In view of the above analysis, the present invention provides a kit and a staining method for special staining of a kidney-puncture tissue biopsy lens, which comprises PASM sets of MASSON stain, MASSON stain and congo red stain, and is used for solving the problems that the existing special staining kit of a kidney-puncture tissue biopsy lens is insufficient in staining a target substance and excessive in staining a non-target substance.
The aim of the invention is mainly realized by the following technical scheme:
The invention provides a kit for special staining of a kidney puncture tissue biopsy lens, which comprises PASM sets of MASSN (mas SON) staining agent, MASSN staining agent and Congo red staining agent;
The PASM sets of MASSON colorants include: periodic acid, hexamine silver working solution, sodium thiosulfate solution, azure blue solution, mayer hematoxylin, lithium carbonate solution, first self-made reagent, second self-made reagent and third self-made reagent;
the MASSON stain comprises a potassium dichromate mixed solution, an azure blue solution, mayer hematoxylin, a lithium carbonate solution, a first self-made reagent, a second self-made reagent and a third self-made reagent;
the Congo red stain comprises Congo red stain solution, potassium hydroxide solution, harris hematoxylin, hydrochloric acid alcohol mixed solution and lithium carbonate solution;
The first homemade reagent comprises bisacodyl red, acid fuchsin, deionized water and glacial acetic acid;
the second self-made reagent comprises phosphomolybdic acid, phosphotungstic acid and deionized water;
The third self-made reagent comprises water-soluble aniline blue, deionized water and glacial acetic acid.
Further, in the PASM sets of MASSON staining agents, the mass concentration of the periodic acid is 0.5-5%;
The hexamine silver working solution comprises hexamethylenetetramine, silver nitrate and sodium tetraborate, wherein the volume ratio of the hexamethylenetetramine to the silver nitrate to the sodium tetraborate is 25:5:3;
The mass concentration of the sodium thiosulfate solution is 2-10%;
The mass concentration of the lithium carbonate solution is 1-10%.
Further, in the MASSON stain, the potassium dichromate mixed solution comprises a potassium dichromate solution and a trichloroacetic acid solution, and the volume ratio of the potassium dichromate solution to the trichloroacetic acid solution is 1:1;
the azure stone blue solution comprises sulfuric acid Gao Tiean, azure stone blue, glycerol and deionized water;
The mass concentration of the lithium carbonate solution is 1-10%.
Further, in the Congo red stain, the Congo red stain is prepared from 0.1-5g of Congo red powder by using 50% ethanol to fix the volume to 100 mL;
the potassium hydroxide solution is prepared by 0.02-2g of potassium hydroxide with 80% ethanol to 100 mL;
The hydrochloric acid-alcohol mixed solution is prepared by adding 99mL of 70% ethanol into 1mL of concentrated hydrochloric acid.
Further, the first self-made reagent is prepared by uniformly mixing 0.4-1.8g of brichimpanzee and 0.05-0.2g of acid fuchsin with deionized water to 100mL of deionized water, and adding 1mL of glacial acetic acid.
Further, the second self-made reagent is prepared by adding deionized water to 1-5g of phosphomolybdic acid and 0.5-5g of phosphotungstic acid to a volume of 100 mL.
Further, the third self-made reagent is prepared by adding deionized water into 1-8g of water-soluble aniline blue to fix the volume to 100mL, and adding 1mL of glacial acetic acid to mix uniformly.
The invention also provides a staining method for special staining of the kidney puncture tissue biopsy lens, which comprises the steps of using PASM sets of MASSN (mas SON) staining agent, MASSN staining agent and Congo red staining agent in the kit to stain different sections of kidney puncture tissue;
The dyeing method using PASM sets of MASSON dyes comprises the following steps: embedding kidney puncture tissue paraffin, preparing a2 mu m paraffin slice, pasting the paraffin slice on a cationic anti-slip sheet, baking the slice, dewaxing and washing the slice, soaking the slice in a 0.5-5% periodate solution for 15-25min, washing the slice for three times, soaking the slice in a hexamine silver working solution, heating the slice in a water bath at 66-86 ℃ for 8-16min, performing microscopic examination, washing the slice for three times, fixing the slice for 1-2min by a sodium thiosulfate solution, washing the slice for three times, soaking the slice in a azure solution for 8-15min, washing the slice for three times, soaking the slice in Mayer hematoxylin for 8-15min, washing the slice for three times, returning the lithium carbonate to blue for several seconds, washing the slice for three times, soaking the slice in a first self-made reagent for 15-25min, washing the slice for three times, soaking the slice in a second self-made reagent for several seconds, soaking the slice in a third self-made reagent for several seconds, washing the slice for three times, and dehydrating the slice.
Further, the dyeing method using the MASSON dye comprises the following steps: after embedding kidney puncture tissue paraffin, preparing a2 mu m paraffin slice, pasting the slice on a cationic anti-slip sheet, baking the slice, dewaxing the slice, directly immersing the slice in BOUIN' S solution without water washing, heating the slice to 55-65 ℃ in a water bath, heating the slice for 40-60min, washing the slice for three times, immersing the slice in potassium dichromate solution for 15-25min, washing the slice for three times, immersing the slice in azure stone blue solution for 8-15min, washing the slice for three times, immersing the slice in Mayer hematoxylin for 8-15min, washing the slice for three times, returning the blue of lithium carbonate for several seconds, washing the slice for three times, immersing the slice in a first self-made reagent for 15-20min, washing the slice for three times, immersing the slice in a second self-made reagent for several seconds, immersing the slice in a third self-made reagent for three times, and dehydrating the slice.
Further, the dyeing method using Congo red dye comprises the following steps: after embedding kidney puncture tissue paraffin, preparing a2 mu m paraffin slice, attaching the paraffin slice onto a cation anti-slip sheet, baking the slice, dewaxing the slice without washing, directly immersing in Congo red dye solution for 5-20min, immersing in potassium hydroxide solution for differentiating for several seconds, washing for three times, immersing in Harris hematoxylin for 8-15min, washing for three times, differentiating for several seconds in hydrochloric acid-alcohol mixed solution, washing for three times, returning lithium carbonate for several seconds, washing for three times, and dehydrating and sealing the slice.
Compared with the prior art, the invention has at least one of the following beneficial effects:
1. The kit for special staining of the kidney puncture tissue biopsy lens comprises three staining agents, proper staining of target substances can be realized through selection of composition materials and accurate design of proportion, and meanwhile, the staining of non-target substances is lighter, the contrast is not excessive, the pathological change position is highlighted, the diagnosis and treatment requirements can be fully met, and the defect situation of the kidney special pathology lens staining kit is compensated.
2. The kit for special staining of the kidney puncture tissue biopsy lens can meet the requirement of simultaneous staining of a maximum of 96 patient kidney puncture tissues in a single staining process, and each kidney puncture tissue in the single staining process has the same staining effect and can meet the requirement of the staining output and quality.
3. The kit and the dyeing method for special dyeing of the kidney puncture tissue biopsy lens can simultaneously finish PASM sets of special dyeing of MASSON, MASSON and Congo red dyeing in a short time, and reduce the labor cost; when the kit disclosed by the invention is used for dyeing kidney puncture tissue, all dyeing processes are completed by only one person, the operation is simple and convenient, no complex operation process is needed, the dyeing process is simple, the dyeing effect is better than that of the existing kit, and the labor and the consumables are saved; meanwhile, the dyeing process is easy to control, and the dyeing process can be stopped at any time.
In the invention, the technical schemes can be mutually combined to realize more preferable combination schemes. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and drawings.
Drawings
The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, like reference numerals being used to designate like parts throughout the drawings;
FIG. 1a is a graph showing the result of staining kidney-penetrating tissue of a patient suffering from kidney disease in example 1 by a kit of the present invention;
FIG. 1b is a graph showing the result of staining kidney-penetrating tissue of a patient with kidney disease in example 1 by an existing kit in a hospital;
FIG. 1c is a graph showing the result of staining kidney-penetrating tissue of a patient suffering from kidney disease in example 2 by a kit of the present invention;
FIG. 1d is a graph showing the result of staining kidney-penetrating tissue of a patient with kidney disease in example 2 by an existing kit in a hospital;
FIG. 2a is a graph showing the result of staining kidney-penetrating tissue of a patient suffering from kidney disease in example 3 by a kit of the present invention;
FIG. 2b is a graph showing the result of staining kidney-penetrating tissue of a patient with kidney disease in example 3 with Solarbio kit;
FIG. 2c is a graph showing the result of staining kidney-penetrating tissue from a remote kit in a renal patient in example 3;
FIG. 3a is a graph showing the result of staining kidney-penetrating tissue of a patient suffering from kidney disease in example 4 by a kit of the present invention;
FIG. 3b is a graph showing the result of staining kidney-penetrating tissue of a patient with kidney disease in example 4 with Solarbio kit;
FIG. 3c is a graph showing the result of staining kidney-penetrating tissue from a remote kit in a renal patient in example 4;
FIG. 4a is a graph showing the result of staining kidney-penetrating tissue of a patient suffering from kidney disease in example 5 by a kit of the present invention;
FIG. 4b is a graph showing the result of staining kidney-penetrating tissue of a patient with kidney disease in example 5 with Solarbio kit;
FIG. 4c is a graph showing the result of staining kidney-penetrating tissue from a remote kit in a renal patient in example 5;
FIG. 5a is a graph showing the result of staining kidney-penetrating tissue of a patient suffering from kidney disease in example 6 by a kit of the present invention;
FIG. 5b is a graph showing the result of staining kidney-penetrating tissue of a patient with kidney disease in example 6 with Solarbio kit;
FIG. 5c is a graph showing the result of staining kidney-penetrating tissue of a patient with kidney disease with a zeup She Shiji box in example 6;
FIG. 6a is a graph showing the result of staining kidney-penetrating tissue of a patient suffering from kidney disease in example 7 by a kit of the present invention;
FIG. 6b is a graph showing the result of staining kidney-penetrating tissue of a patient with kidney disease in example 7 with Solarbio kit;
FIG. 6c is a graph showing the result of staining kidney-penetrating tissue of a patient suffering from kidney disease in example 7 with LBP kit.
Detailed Description
The following detailed description of preferred embodiments of the application is made in connection with the accompanying drawings, which form a part hereof, and together with the description of the embodiments of the application, are used to explain the principles of the application and are not intended to limit the scope of the application.
The invention provides a kit for special staining of a kidney puncture tissue biopsy lens, which comprises PASM sets of MASSN (master batch, automatic teller machine) staining agent, MASSN staining agent and Congo red staining agent;
The PASM sets of MASSON colorants include: periodic acid, hexamine silver working solution, sodium thiosulfate solution, azure blue solution, mayer hematoxylin, lithium carbonate solution, first self-made reagent, second self-made reagent and third self-made reagent;
the MASSON stain comprises a potassium dichromate mixed solution, an azure blue solution, mayer hematoxylin, a lithium carbonate solution, a first self-made reagent, a second self-made reagent and a third self-made reagent;
the Congo red stain comprises Congo red stain solution, potassium hydroxide solution, harris hematoxylin, hydrochloric acid alcohol mixed solution and lithium carbonate solution.
The PASM sets of MASSON colorants and the MASSON colorants each include a first self-made reagent, a second self-made reagent, and a third self-made reagent. In PASM sets of MASSON stain, the first homemade reagent has the same composition, but may have the same or different composition contents.
Specifically, the mass concentration of the periodic acid in PASM sets of MASSON staining agent is 0.5-5%, and the MASSON staining agent is prepared by using deionized water to fix the volume of 0.5-5 g of periodic acid to 100 mL;
the hexamine silver working solution is prepared from hexamethylenetetramine, silver nitrate and sodium tetraborate according to the mass ratio of 25:5:3;
PASM sets of MASSON stain, wherein the mass concentration of hexamethylenetetramine is 1-6%, and the MASSON stain is prepared by using 1-6g of hexamethylenetetramine to fix the volume to 100mL by deionized water; the mass concentration of the silver nitrate is 1-5%, and the silver nitrate is prepared by 1-5g of silver nitrate with deionized water to 100 mL; the sodium tetraborate has the mass concentration of 2-10%, and is prepared by dissolving 2-10g of borax in deionized water at 70 ℃ and then fixing the volume to 100 mL;
The mass concentration of the sodium thiosulfate solution is 2-10%, and the sodium thiosulfate solution is prepared by using 2-10g of sodium thiosulfate to reach 100mL with deionized water to fix the volume;
The mass concentration of the azure solution is 2-10%, 2-10g of sulfuric acid Gao Tiean and 1g of azure are added with more than 20mL of deionized water to be boiled, the volume is fixed to 200mL, 30mL of glycerin is added to be uniformly mixed, and the mixture is stored at 4 ℃;
the mass concentration of the lithium carbonate solution is 1-10%, and 1-10g of lithium carbonate is added with deionized water to a volume of 100mL to prepare the lithium carbonate solution;
The first self-made reagent is prepared by uniformly mixing 0.4-1.8g of Bifiduciary scarlet and 0.05-0.2g of acid fuchsin with deionized water to 100mL of volume, and adding 1mL of glacial acetic acid; the reagent can strengthen the color of the eosinophilic hemoglobin, so that immune complex deposition bright red of IgA nephropathy, membranous nephropathy and lupus nephritis is more obvious, is obviously different from rose red of renal tubular epithelial cell cytoplasm, and provides basis for judging IgA nephropathy, membranous nephropathy and lupus nephritis;
The second self-made reagent is prepared by adding deionized water into 1-5g of phosphomolybdic acid and 0.5-2g of phosphotungstic acid, and fixing the volume to 100 mL; the reagent can differentiate the dyeing effect of the first self-made reagent, weaken the dyeing of non-target substances and highlight the dyeing effect of the target substances; meanwhile, the collagen can be used as an intermediate coupling substance of a third self-made reagent, so that the collagen can be better combined with the type III collagen;
The third self-made reagent is prepared by adding deionized water into 1-5g of water-soluble aniline blue to a volume of 100mL, adding 1mL of glacial acetic acid, and uniformly mixing; the reagent can be combined with a second self-made reagent, so that the collagen III presents blue color;
It should be noted that PASM sets of MASSON colorants in existing commercial kits typically include an oxidizing agent, an iron alum solution, a hexamine silver working solution, a gold chloride solution, a hypotonic solution, and a light green solution. Compared with PASM sets of MASSON staining agents in the existing commercial kit, the PASM sets of MASSON staining agents in the invention have different formulas of periodic acid and hexamine silver working solution, and do not comprise ferric alum solution, gold chloride solution, hypotonic solution and light green solution; the PASM sets of MASSON staining agent provided by the invention can clearly observe and locate glomerular basement membrane lesions (such as whether ribbon-like changes, spike, chain ring, double-track diseases and mesangial insertions exist) through interaction of the constituent reagents.
Specifically, in the MASSON stain, the potassium dichromate mixed solution is formed by mixing a potassium dichromate solution and a trichloroacetic acid solution according to the proportion of 1:1, wherein the potassium dichromate solution is prepared by adding deionized water into 50-200g of potassium dichromate to a volume of 1000mL, and the trichloroacetic acid solution is prepared by adding deionized water into 50-200g of trichloroacetic acid to a volume of 1000 mL;
the azure blue solution is prepared by adding more than 20mL of deionized water into 1-20g of sulfuric acid Gao Tiean and 0.5-5g of azure blue, boiling, fixing the volume to 200mL, adding 30mL of glycerol, uniformly mixing, and preserving at 4 ℃;
the mass concentration of the lithium carbonate solution is 1-10%, and 1-10g of lithium carbonate is added with deionized water to a volume of 100mL to prepare the lithium carbonate solution;
The first self-made reagent is prepared by uniformly mixing 0.4-1.8g of Bifiduciary scarlet and 0.05-0.2g of acid fuchsin with deionized water to 100mL of volume, and adding 1mL of glacial acetic acid;
The second self-made reagent is prepared by adding deionized water into 1-5g of phosphomolybdic acid and 0.5-5g of phosphotungstic acid to a volume of 100 mL;
the third self-made reagent is prepared by adding deionized water into 1-8g of water-soluble aniline blue to a volume of 100mL, adding 1mL of glacial acetic acid, and uniformly mixing;
It should be noted that PASM sets of MASSON dyes in the existing commercial kit generally include azure solution, mayer hematoxylin, acidic ethanol differentiation solution, ponceau dye solution, phosphomolybdic acid solution, and weak acid solution. Compared with the MASSON staining agent in the existing commercial kit, the MASSON staining agent does not comprise an acidic ethanol differentiation solution and a weak acid solution; the first self-made reagent is not a single ponceau solution, but a mixed solution of brichimpanzee, acid fuchsin and glacial acetic acid; the second self-made reagent is not a single phosphomolybdic acid solution, but a mixed solution of phosphomolybdic acid and phosphotungstic acid; meanwhile, a potassium dichromate mixed solution, a lithium carbonate solution and a third self-made reagent are added. By interaction of the constituent reagents, the MASSON stain of the present invention can clearly observe and localize the deposition amount, deposition site and renal interstitial fibrosis of the eosinophilic hemoglobin.
It should be noted that, the concentrations of the second self-made reagent and the third self-made reagent in PASM sets of MASSON staining agent and MASSON staining agent are different, because PASM sets of MASSON staining agent and MASSON staining agent are different in purpose, PASM sets of MASSON staining agent mainly observe glomerular basement membrane lesions (such as whether ribbon-like changes, nailing, chain ring, double rail diseases and tie membrane insertion exist) and the tie membrane region and the basement membrane staining are black, so that the red color of the reddish philin is blocked and specific deposition part of the protein cannot be observed; the purpose of MASSON staining is to observe the deposition amount, deposition site and renal interstitial fibrosis of the basophilic rhodopsin, the glomerular basement membrane and the renal interstitial fibrosis are rich in collagen type three, the high concentration of the second homemade reagent and the third homemade reagent can enable the staining agent to be more tightly combined with the collagen type three, the bason and the deposition amount thereof are better positioned, the red-blue contrast is high, the differentiation is easier under a microscope, the blue duty ratio of the renal interstitial is judged to be more clear by the high concentration of the second homemade reagent and the third homemade reagent, and therefore, the concentration of the second homemade reagent and the third homemade reagent in the MASSON staining agent is slightly higher.
Specifically, in Congo red stain, congo red stain is prepared from 0.1-5g of Congo red powder to 100mL by 50% ethanol; the potassium hydroxide solution is prepared by adding 0.02-2g of potassium hydroxide into 80% ethanol to a volume of 100mL, and adding 99mL of 70% ethanol into 1mL of concentrated hydrochloric acid to prepare a hydrochloric acid-alcohol mixed solution; the mass concentration of the lithium carbonate solution is 1-10%, and the lithium carbonate solution is prepared by adding 1-10g of lithium carbonate into deionized water to fix the volume to 100 mL.
Through the kit, different sections of kidney puncture tissues can be dyed, and dewaxed and washed 2 mu m paraffin sections can be dyed through PASM sets of MASSON staining agents, wherein the dyeing method comprises the following steps: embedding kidney puncture tissue paraffin, preparing a2 mu m paraffin slice, pasting the paraffin slice on a cationic anti-slip sheet, baking the slice, dewaxing and washing the slice, soaking the slice in a 0.5-5% periodate solution for 15-25min, washing the slice for three times, soaking the slice in a hexamine silver working solution, heating the slice in a water bath at 66-86 ℃ for 8-16min, performing microscopic examination, washing the slice for three times, fixing the slice for 1-2min by a sodium thiosulfate solution, washing the slice for three times, soaking the slice in a azure solution for 8-15min, washing the slice for three times, soaking the slice in Mayer hematoxylin for 8-15min, washing the slice for three times, returning the lithium carbonate to blue for several seconds, washing the slice for three times, soaking the slice in a first self-made reagent for 15-25min, washing the slice for three times, soaking the slice in a second self-made reagent for several seconds, soaking the slice in a third self-made reagent for several seconds, washing the slice for three times, and dehydrating the slice.
The dewaxed and washed 2 mu m paraffin section can be dyed by MASSON staining agent, and the dyeing method comprises the following steps: after embedding kidney puncture tissue paraffin, preparing a 2 mu m paraffin slice, pasting the slice on a cationic anti-slip sheet, baking the slice, dewaxing the slice, directly immersing the slice in BOUIN' S solution without water washing, heating the slice to 55-65 ℃ in a water bath, heating the slice for 40-60min, washing the slice for three times, immersing the slice in potassium dichromate solution for 15-25min, washing the slice for three times, immersing the slice in azure stone blue solution for 8-15min, washing the slice for three times, immersing the slice in Mayer hematoxylin for 8-15min, washing the slice for three times, returning the blue of lithium carbonate for several seconds, washing the slice for three times, immersing the slice in a first self-made reagent for 15-20min, washing the slice for three times, immersing the slice in a second self-made reagent for several seconds, immersing the slice in a third self-made reagent for three times, and dehydrating the slice.
The dewaxed paraffin section with the diameter of 2 mu m can be dyed by using Congo red coloring agent, and the dyeing method comprises the following steps: after embedding kidney puncture tissue paraffin, preparing a2 mu m paraffin slice, attaching the paraffin slice onto a cation anti-slip sheet, baking the slice, dewaxing the slice without washing, directly immersing in Congo red dye solution for 5-20min, immersing in potassium hydroxide solution for differentiating for several seconds, washing for three times, immersing in Harris hematoxylin for 8-15min, washing for three times, differentiating for several seconds in hydrochloric acid-alcohol mixed solution, washing for three times, returning lithium carbonate for several seconds, washing for three times, and dehydrating and sealing the slice.
It should be noted that PASM sets of MASSON dye, MASSON dye and congo red dye act on a continuous slice of the same kidney puncture tissue of the same patient to dye respectively, and the three dyes dye all parts of the continuous slice, but the coloring effect is different, and the three dyes respectively highlight different lesions and do not dye the same dyed object in a superposition way.
The kit for special staining of the kidney puncture tissue biopsy lens comprises three staining agents, and through the selection of the composition materials and the accurate design of the proportion, the moderate staining of target substances can be realized, and meanwhile, the staining of non-target substances is lighter, the excessive staining is avoided, and the diagnosis and treatment requirements can be fully met; the single dyeing can meet the simultaneous dyeing of the maximum 96 patient kidney puncture tissues, and each kidney puncture tissue in the single dyeing has the same dyeing effect, so that the dyeing yield and quality can be simultaneously met; meanwhile, the kit can simultaneously complete PASM sets of special dyeing of MASSON, MASSON and Congo red in a short time, so that the labor cost is reduced; when the kit disclosed by the invention is used for dyeing kidney puncture tissue, all dyeing processes are completed by only one person, the operation is simple and convenient, no complex operation process is needed, the dyeing process is simple, the dyeing effect is better than that of the existing kit, and the labor and the consumables are saved; meanwhile, the dyeing process is easy to control, and the dyeing process can be stopped at any time.
Example 1
And staining kidney-penetrating tissue of the patient suffering from the kidney disease by the kit for special staining of the kidney-penetrating tissue biopsy lens.
The kit comprises PASM sets of MASSON stain, MASSON stain and Congo red stain;
The PASM sets of MASSON colorants include: periodic acid, hexamine silver working solution, sodium thiosulfate solution, azure blue solution, mayer hematoxylin, lithium carbonate solution, first self-made reagent, second self-made reagent and third self-made reagent;
the MASSON stain comprises a potassium dichromate mixed solution, an azure blue solution, mayer hematoxylin, a lithium carbonate solution, a first self-made reagent, a second self-made reagent and a third self-made reagent;
the Congo red stain comprises Congo red stain solution, potassium hydroxide solution, harris hematoxylin, hydrochloric acid alcohol mixed solution and lithium carbonate solution.
Wherein, in PASM sets of MASSON staining agent, the mass concentration of the periodic acid is 3 percent, and the agent is prepared by fixing the volume of 3g of periodic acid to 100mL by deionized water; the hexamine silver working solution is prepared from hexamethylenetetramine, silver nitrate and sodium tetraborate according to the mass ratio of 25:5:3; wherein the mass concentration of the hexamethylenetetramine is 5%, and the hexamethylenetetramine is prepared by 5g of hexamethylenetetramine with deionized water to 100 mL; the mass concentration of the silver nitrate is 5%, and the silver nitrate is prepared by 5g of silver nitrate with deionized water to reach a volume of 100 mL; the sodium tetraborate has the mass concentration of 6%, and is prepared by dissolving 6g of borax in deionized water at 70 ℃ and then fixing the volume to 100 mL; the mass concentration of the sodium thiosulfate solution is 6%, and the sodium thiosulfate solution is prepared by 6g of sodium thiosulfate with deionized water to reach a volume of 100 mL; the mass concentration of the azure blue solution is 5%, 5g of sulfuric acid Gao Tiean and 1g of azure blue are added with more than 20mL of deionized water to be boiled, the volume is fixed to 200mL, 30mL of glycerol is added to be uniformly mixed, and the mixture is stored at 4 ℃; the mass concentration of the lithium carbonate solution is 5%, and 5g of lithium carbonate is added with deionized water to fix the volume to 100 mL; the first self-made reagent is prepared by uniformly mixing 1.5g of Bifiduciary scarlet and 0.1g of acid fuchsin with deionized water to 100mL and adding 1mL of glacial acetic acid; the second self-made reagent is prepared by adding deionized water into 3g of phosphomolybdic acid and 1.0g of phosphotungstic acid, and fixing the volume to 100 mL; the third self-made reagent is prepared by adding deionized water into 3g of water-soluble aniline blue to fix the volume to 100mL, and adding 1mL of glacial acetic acid to mix uniformly.
Wherein, in the MASSON coloring agent, the mixed solution of potassium dichromate is formed by mixing a potassium dichromate solution and a trichloroacetic acid solution according to the proportion of 1:1, wherein the potassium dichromate solution is prepared by adding deionized water into 100g of potassium dichromate to a volume of 1000mL, and the trichloroacetic acid solution is prepared by adding deionized water into 100g of trichloroacetic acid to a volume of 1000 mL; the azure blue solution is prepared by adding more than 20mL of deionized water into 10g of sulfuric acid Gao Tiean and 3.0g of azure blue, boiling, fixing the volume to 200mL, adding 30mL of glycerol, uniformly mixing, and preserving at 4 ℃; the mass concentration of the lithium carbonate solution is 5%, and 5g of lithium carbonate is added with deionized water to fix the volume to 100 mL; the first self-made reagent is prepared by uniformly mixing 1.5g of Bifiduciary scarlet and 0.1g of acid fuchsin with deionized water to 100mL and adding 1mL of glacial acetic acid; the second self-made reagent is prepared by adding deionized water into 5.0g of phosphomolybdic acid and 3.0g of phosphotungstic acid to a volume of 100 mL; the third self-made reagent is prepared by adding deionized water into 6g of water-soluble aniline blue to fix the volume to 100mL, and adding 1mL of glacial acetic acid to mix uniformly.
Wherein, in the Congo red coloring agent, 3.0g of Congo red powder is used for fixing the volume of the Congo red coloring liquid to 100mL by 50% ethanol; the potassium hydroxide solution is prepared by adding 99mL of 70% ethanol into 1mL of concentrated hydrochloric acid, wherein 1.0g of potassium hydroxide is fixed to 100mL of 80% ethanol; the mass concentration of the lithium carbonate solution is 5.0%, and 5g of lithium carbonate is added with deionized water to fix the volume to 100 mL.
The kidney puncture tissue of a patient suffering from kidney disease is stained by PASM sets of MASSON staining agent, and the staining method comprises the following steps: embedding kidney puncture tissue paraffin of a patient suffering from kidney disease, preparing a2 mu m paraffin slice, pasting the slice on a cation anti-dislocation slice, dewaxing and washing the slice, soaking the slice in a 3% periodate solution for 17min, washing the slice for three times, soaking the slice in a hexamine silver working solution, heating the slice in a water bath at 76 ℃ for 10min, performing microscopic examination, washing the slice for three times, fixing the slice in sodium thiosulfate for 1min, washing the slice for three times, soaking the slice in a azure solution for 10min, washing the slice for three times, soaking the slice in Mayer hematoxylin for 10min, washing the slice for three times, returning lithium carbonate for 10s, washing the slice for three times, soaking the slice in a first self-made reagent for 17min, washing the slice for three times, soaking the slice in a second self-made reagent for 10s, soaking the slice in a third self-made reagent for 15s, washing the slice for three times, and dehydrating the slice.
Staining kidney puncture tissue of a patient suffering from kidney disease by using a MASSON staining agent, wherein the staining method comprises the following steps: after embedding kidney puncture tissue paraffin of a kidney disease patient, preparing a2 mu m paraffin slice, pasting the paraffin slice on a cation anti-slip sheet, baking the slice, dewaxing without washing, directly immersing the slice in BOUIN' S solution, heating the slice to 62 ℃ in a water bath, heating the slice for 60min, washing the slice for three times, immersing the slice in potassium dichromate solution for 20min, washing the slice for three times, immersing the slice in azure solution for 10min, washing the slice for three times, immersing the slice in Mayer hematoxylin for 10min, washing the slice for three times, returning lithium carbonate to blue for 10S, washing the slice for three times, immersing the slice in a first self-made reagent for 17min, washing the slice for three times, immersing the slice in a second self-made reagent for 10S, immersing the slice in a third self-made reagent for 15S, washing the slice for three times, and dehydrating the slice.
The renal puncture tissue of a patient suffering from the kidney disease is stained by using Congo red stain, and the staining method comprises the following steps: after embedding kidney puncture tissue paraffin of a kidney disease patient, preparing a2 mu m paraffin slice, pasting the paraffin slice on a cation anti-slip sheet, dewaxing after baking the slice, directly immersing in Congo red dye solution for 15min, immersing in potassium hydroxide solution for differentiating for 4s, washing for three times, immersing in Harris hematoxylin for 10min, washing for three times, differentiating in hydrochloric acid-alcohol mixed solution for 10s, washing for three times, returning lithium carbonate for 10s, washing for three times, and dehydrating and sealing the slice.
FIG. 1a is a graph showing that kidney-penetrating tissue of a patient suffering from kidney disease in example 1 was stained with the kit, and it can be seen from the graph that glomerular basement membrane typical spike (part of burr) is recognized as stage II membranous kidney disease.
Comparative example 1
Kidney-penetrating tissue from renal patients in example 1 was stained by an existing kit from a hospital (zilu medical laboratory).
The dyeing method is the same as in example 1.
FIG. 1b is a diagram showing the kidney-penetrating tissue of a patient suffering from kidney disease in example 1, which is stained with an existing kit from a hospital (Qilu medical test institute), and the stained kidney-penetrating tissue is too dense to clearly observe whether there is spike on glomerular basement membrane, which makes pathological diagnosis difficult, requires repeated staining, increases diagnosis time, and loses kidney-penetrating tissue; it is not possible to confirm whether the patient is a stage II membranous nephropathy patient.
Example 2
And staining kidney-penetrating tissue of the patient suffering from the kidney disease by the kit for special staining of the kidney-penetrating tissue biopsy lens.
The dyeing method is the same as in example 1.
FIG. 1c is a graph showing staining of kidney-penetrating tissue from a patient with kidney disease by the kit, and from the graph, it can be seen that the glomerular basement membrane is typically altered in a ribbon-like fashion (translucent broadening), which can be used to determine that the patient is stage I membranous kidney disease.
Comparative example 2
Kidney-penetrating tissue from renal patients in example 2 was stained by an existing kit from a hospital (first hospital in the university of lan).
The dyeing method is the same as in example 1.
FIG. 1d is a graph showing that the kidney-penetrating tissue of a patient suffering from kidney disease in example 2 is stained by the conventional kit, and it can be seen from the graph that the stained kidney-penetrating tissue is too pale, and the change of the glomerular basement membrane ribbon pattern cannot be clearly observed, which hinders pathological diagnosis, requires repeated staining, increases diagnosis time, and loses kidney-penetrating tissue; it is impossible to confirm whether the patient is a patient with stage I membranous nephropathy.
Example 3
The kit for special staining of the kidney puncture tissue biopsy lens is used for staining the kidney puncture tissue of a patient with certain fibrous glomerulus.
The dyeing method is the same as in example 1.
FIG. 2a shows the patient's kidney-penetrating tissue stained by the kit, and the enlargement of the mesangial region, thickening of the basement membrane accompanied by specific protein deposition (grey part of the figure) is clearly observed.
Comparative example 3
The kidney-penetrating tissue of a patient with fibrous glomerular disease in example 3 was stained by an existing Solarbio kit.
The dyeing method is the same as in example 1.
FIG. 2b shows the patient's kidney-penetrating tissue stained with Solarbio kit, where the staining is overall heavy and it is indistinguishable whether a specific protein is deposited.
Comparative example 4
The kidney-penetrating tissue of a certain fibrous glomerulopathy patient in example 3 was stained by means of an existing far mu kit.
The dyeing method is the same as in example 1.
Fig. 2c shows the patient's kidney-penetrating tissue stained with the far mu kit, and it can be seen that the whole staining is too shallow, and it is not only impossible to distinguish the pathological conditions of the basement membrane and the peri-membrane region, but also impossible to determine whether there is immune complex deposition.
Example 4
And staining the kidney puncture tissue of a patient suffering from the II-stage membranous nephropathy by using the kit for special staining of the kidney puncture tissue biopsy lens.
The dyeing method is the same as in example 1.
Fig. 3a shows the staining of kidney-penetrating tissue of the patient by the kit, from which basal membrane spike is clearly visible.
Comparative example 5
The kidney-penetrating tissue of a patient with stage II membranous nephropathy in example 4 was stained by an existing Solarbio kit.
The dyeing method is the same as in example 1.
FIG. 3b shows the patient's kidney-penetrating tissue stained with Solarbio kit, from which no spike is visible.
Comparative example 6
Kidney-penetrating tissue from a patient with stage ii membranous nephropathy in example 4 was stained by means of an existing far mu kit.
The dyeing method is the same as in example 1.
FIG. 3c shows staining of kidney-penetrating tissue of the patient with a remote mu kit, with no spike visible from the figure; in pathological diagnosis, only fig. 3a of example 4 can be diagnosed as stage ii membranous nephropathy.
Example 5
And staining the kidney puncture tissue of a patient suffering from the phase I membranous nephropathy by using the kit for special staining of the kidney puncture tissue biopsy lens.
The dyeing method is the same as in example 1.
Fig. 4a shows the staining of kidney-penetrating tissue of the patient by the kit, from which a ribbon-like change is clearly observed.
Comparative example 7
Kidney-penetrating tissue from a patient with stage i membranous nephropathy in example 5 was stained by an existing Solarbio kit.
The dyeing method is the same as in example 1.
FIG. 4b shows that the patient's kidney-penetrating tissue was stained with Solarbio kit, and the whole staining was thick and was not clearly typed.
Comparative example 8
Kidney-penetrating tissue from a patient with stage i membranous nephropathy in example 5 was stained by means of an existing far mu kit.
The dyeing method is the same as in example 1.
FIG. 4c shows the patient's kidney-penetrating tissue stained with a far mu kit, as can be seen from the figure, the stained whole is too shallow to be clearly typed; in pathological diagnosis, only FIG. 4a can be diagnosed as stage I membranous nephropathy.
Example 6
And (3) staining the kidney puncture tissue of a patient suffering from lupus nephritis by using the kit for special staining of the kidney puncture tissue biopsy lens.
The dyeing method is the same as in example 1.
Fig. 5a shows that the kidney puncture tissue of the patient is stained by the kit, the deposition of the eosinophilic hemoglobin can be obviously observed from the graph, and the glomerular bowman's capsule and the tubular basement membrane are obviously outlined by blue lines, so that different areas can be obviously divided.
Comparative example 9
The kidney-punctured tissue of lupus nephritis patients in example 6 was stained by the existing Solarbio kit.
The dyeing method is the same as in example 1.
FIG. 5b is a graph of the patient's kidney-penetrating tissue stained with Solarbio kit, where it can be seen that the red coloration is insufficient and no significant immune complex deposition is observed, but blue-stained type three collagen is visible.
Comparative example 10
The kidney-punctured tissue of lupus nephritis patients in example 6 was stained by an existing ze She Shiji cassette.
The dyeing method is the same as in example 1.
FIG. 5c shows the patient's kidney-penetrating tissue stained with the She Shiji box, and it can be seen that the overall coloration is insufficient and that neither immune complex nor type III collagen is clearly visible.
Example 7
Staining kidney-penetrating tissue of a patient with a type-2 LECT amyloidosis by means of the kit for kidney-penetrating tissue biopsy.
The dyeing method is the same as in example 1.
FIG. 6a shows the patient's kidney-penetrating tissue stained by the kit, from which the positive parts are visibly stained in a dark, bright red, distinguishable from the non-specific background.
Comparative example 11
Kidney-penetrating tissue from a patient suffering from an amyloidosis type LECT2 of example 7 was stained by means of the existing Solarbio kit.
The dyeing method is the same as in example 1.
FIG. 6b shows the patient's kidney-penetrating tissue stained with Solarbio kit, where it can be seen that the non-specific background and positive sites are not clearly distinguishable.
Comparative example 12
The kidney-punctured tissue of lupus nephritis patients in example 7 was stained by the existing LBP kit.
The dyeing method is the same as in example 1.
FIG. 6c shows staining of kidney-penetrating tissue of the patient with LBP kit, where it can be seen that non-specific background and positive sites are indistinguishable.
According to the embodiment and the comparative example, the kit provided by the invention comprises three kinds of coloring agents, and through the selection of the composition materials and the accurate design of the proportion, the proper coloring of target substances can be realized, meanwhile, the coloring of non-target substances is lighter, the coloring is not excessive, and the dyeing effect is better than that of the existing kit.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (10)
1. A kit for special staining of a kidney-penetrating tissue biopsy lens, comprising PASM sets of MASSON stain, and congo red stain;
The PASM sets of MASSON colorants include: periodic acid, hexamine silver working solution, sodium thiosulfate solution, azure blue solution, mayer hematoxylin, lithium carbonate solution, first self-made reagent, second self-made reagent and third self-made reagent;
the MASSON stain comprises a potassium dichromate mixed solution, an azure blue solution, mayer hematoxylin, a lithium carbonate solution, a first self-made reagent, a second self-made reagent and a third self-made reagent;
the Congo red stain comprises Congo red stain solution, potassium hydroxide solution, harris hematoxylin, hydrochloric acid alcohol mixed solution and lithium carbonate solution;
The first homemade reagent comprises bisacodyl red, acid fuchsin, deionized water and glacial acetic acid;
the second self-made reagent comprises phosphomolybdic acid, phosphotungstic acid and deionized water;
The third self-made reagent comprises water-soluble aniline blue, deionized water and glacial acetic acid.
2. The kit according to claim 1, wherein the mass concentration of periodic acid in the PASM sets of MASSON colorants is 0.5-5%;
The hexamine silver working solution comprises hexamethylenetetramine, silver nitrate and sodium tetraborate, wherein the volume ratio of the hexamethylenetetramine to the silver nitrate to the sodium tetraborate is 25:5:3;
The mass concentration of the sodium thiosulfate solution is 2-10%;
The mass concentration of the lithium carbonate solution is 1-10%.
3. The kit according to claim 2, wherein in the MASSON stain, the mixed solution of potassium dichromate comprises a potassium dichromate solution and a trichloroacetic acid solution, and the volume ratio of the potassium dichromate solution to the trichloroacetic acid solution is 1:1;
the azure stone blue solution comprises sulfuric acid Gao Tiean, azure stone blue, glycerol and deionized water;
The mass concentration of the lithium carbonate solution is 1-10%.
4. The kit of claim 3, wherein in the congo red stain, the congo red stain is formulated from 0.1-5g congo red powder to a volume of 50% ethanol to 100 mL;
the potassium hydroxide solution is prepared by 0.02-2g of potassium hydroxide with 80% ethanol to 100 mL;
The hydrochloric acid-alcohol mixed solution is prepared by adding 99mL of 70% ethanol into 1mL of concentrated hydrochloric acid.
5. The kit according to claim 4, wherein the first self-made reagent is prepared by mixing 0.4-1.8g of brichimpanzee and 0.05-0.2g of acid fuchsin with deionized water to a volume of 100mL, and adding 1mL of glacial acetic acid.
6. The kit of claim 5, wherein the second self-made reagent is prepared by adding 1-5g phosphomolybdic acid and 0.5-5g phosphotungstic acid to deionized water to a volume of 100 mL.
7. The kit according to claim 5, wherein the third self-made reagent is prepared by adding deionized water to 1-8g of water-soluble aniline blue to a volume of 100mL, and adding 1mL of glacial acetic acid to mix uniformly.
8. A staining method for special staining of a kidney-penetrating tissue biopsy lens by the kit of any of claims 1-7, comprising staining different sections of kidney-penetrating tissue with PASM sets of MASSON stain, MASSON stain and congo red stain in the kit;
The dyeing method using PASM sets of MASSON dyes comprises the following steps: embedding kidney puncture tissue paraffin, preparing a2 mu m paraffin slice, pasting the paraffin slice on a cationic anti-slip sheet, baking the slice, dewaxing and washing the slice, soaking the slice in a 0.5-5% periodate solution for 15-25min, washing the slice for three times, soaking the slice in a hexamine silver working solution, heating the slice in a water bath at 66-86 ℃ for 8-16min, performing microscopic examination, washing the slice for three times, fixing the slice for 1-2min by a sodium thiosulfate solution, washing the slice for three times, soaking the slice in a azure solution for 8-15min, washing the slice for three times, soaking the slice in Mayer hematoxylin for 8-15min, washing the slice for three times, returning the lithium carbonate to blue for several seconds, washing the slice for three times, soaking the slice in a first self-made reagent for 15-25min, washing the slice for three times, soaking the slice in a second self-made reagent for several seconds, soaking the slice in a third self-made reagent for several seconds, washing the slice for three times, and dehydrating the slice.
9. The method of dyeing according to claim 8, wherein the method of dyeing using MASSON colorants is: after embedding kidney puncture tissue paraffin, preparing a2 mu m paraffin slice, pasting the slice on a cationic anti-slip sheet, baking the slice, dewaxing the slice, directly immersing the slice in BOUIN' S solution without water washing, heating the slice to 55-65 ℃ in a water bath, heating the slice for 40-60min, washing the slice for three times, immersing the slice in potassium dichromate solution for 15-25min, washing the slice for three times, immersing the slice in azure stone blue solution for 8-15min, washing the slice for three times, immersing the slice in Mayer hematoxylin for 8-15min, washing the slice for three times, returning the blue of lithium carbonate for several seconds, washing the slice for three times, immersing the slice in a first self-made reagent for 15-20min, washing the slice for three times, immersing the slice in a second self-made reagent for several seconds, immersing the slice in a third self-made reagent for three times, and dehydrating the slice.
10. The method of dyeing according to claim 9, wherein the method of dyeing with congo red dye is: after embedding kidney puncture tissue paraffin, preparing a2 mu m paraffin slice, attaching the paraffin slice onto a cation anti-slip sheet, baking the slice, dewaxing the slice without washing, directly immersing in Congo red dye solution for 5-20min, immersing in potassium hydroxide solution for differentiating for several seconds, washing for three times, immersing in Harris hematoxylin for 8-15min, washing for three times, differentiating for several seconds in hydrochloric acid-alcohol mixed solution, washing for three times, returning lithium carbonate for several seconds, washing for three times, and dehydrating and sealing the slice.
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