CN103926131B - The hexamine Yin Masong resisdye method improved - Google Patents

Abstract

The present invention relates to the hexamine Yin Masong resisdye method of a kind of improvement, comprise the following steps: preparation silver transfection reagent; By liquid-solid for tissue slice Bouin fixed; 2.5% iodine tincture, 5% sodium thiosulfate, 1.5% periodic acid are immersed in section successively; Take silver dye A liquid and silver dye B immersion dye section, terminate after black clearly until basement membrane shows; 3% sodium thiosulfate soaks section; Haematoxylin is contaminated; Hydrochloride alcohol breaks up, and indigo plant is returned in washing; Ponceaux dye liquor is contaminated; 1% phosphomolybdic acid soaks section; Dimethylbenzene is entered transparent, neutral gum mounting after gradient alcohol dehydration. The invention provides the allotment of a kind of reagent rationally, Color is good and the time is short, stable performance, resisdye lovely luster hexamine Yin Masong resisdye method, and achieve effect more better than existing method in an experiment, change Silver stain is organized and dye easily occurred, time, the more difficult control of temperature, the problem such as background color is too high and resisdye other dyeing color is bright-coloured not.

Description

The hexamine Yin Masong resisdye method improved

Technical field

The present invention relates to hexamine Yin Masong resisdye method, be one of specific stain method that the pathological diagnosis of Renal biopsy is necessary. Especially, the present invention relates to the hexamine Yin Masong resisdye method of a kind of improvement, it is possible to be used for showing that bead in nephridial tissue, tubule, basement membrane of blood vessel and histogenic immunity complex deposits and fibrosis etc. change.

Background technology

Hexamine Yin Masong resisdye method is the colouring method for nephridial tissue section that the hexamine silver method evolution from mycete dyeing is formed, and is hexamine silver and the superposition dyeing of the loose two kinds of colouring methods of horse. In renal glomerular disease, often resulting in the change of glomerular basement membrane, basement membrane is the lining form of a layer height property of water-bearing gelinite, is mainly made up of mucopolysaccharide and protein, has permeability, is often intimately associated with very thin connective fiber. Hexamine Yin Masong resisdye method is to make nephridial tissue after acid oxidase, and the mucopolysaccharide in basement membrane exposes aldehyde radical, and aldehyde radical is reduced to the argent of pitchy hexamine silver, so that basement membrane demonstrates the painted of black. This method is to be one of conventional specific stain method in renal needle biopsy pathological diagnosis. It can not only clearly show the number of bead in nephridial tissue, sketch out the fracture of bead basement membrane, hypertrophy, the morphologic change such as folding, can also show that immune complex is at basement membrane deposition position exactly, moreover it is possible to demonstrate renal tubules atrophing state, vessel wall thickening whether and the order of severity etc. of cell infiltration. Therefore, hexamine Yin Masong resisdye method provides important foundation for Pathological clinical diagnosis, classification and treatment, is the specific stain method that Pathological diagnosis is essential and difficulty is maximum.

At present, professional person both domestic and external wants that having use up thousand and one way improves conventional basement membrane Silver stain method, including Gomori method, Jone ' s method, vow island (Jenos) improved method, Gordon-Sweet method, Janes method etc., attempt obtains better basement membrane dyeing quality, but still there is following variety of problems: organizes in Silver stain and dye easily occurred; Time, the more difficult control of temperature; Background color is too high; Resisdye other dyeing color is bright-coloured not.

Summary of the invention

It is an object of the invention to provide the hexamine Yin Masong resisdye method of a kind of improvement, silver-colored transfection reagent allotment in the method is rationally, Color is good, the time is short, stable performance, resisdye dyeing lovely luster, it is possible to provide important foundation for Pathological clinical diagnosis, classification and treatment better.

The hexamine Yin Masong resisdye method of a kind of improvement of the present invention, comprises the following steps:

(1) preparation silver transfection reagent, including:

Silver dye A liquid: distilled water 230ml, 5% Borax 45ml, 4% boric acid 4ml, 15% hexamethylenetetramine 60ml; Silver dye B liquid: distilled water 100ml, 10g silver nitrate;

5% Borax: distilled water 100ml, 5g Borax;

15% hexamethylenetetramine: distilled water 100ml, 15g hexamethylenetetramine;

4% boric acid: distilled water 100ml, 4g boric acid;

1.5% periodic acid: distilled water 100ml, 1.5g periodic acid;

Ponceaux dye liquor: Ponceau S 0.7g, acid fuchsin 0.3g, distilled water 100ml, glacial acetic acid 1ml;

1% aniline blue: aniline blue 1g, glacial acetic acid 1ml, distilled water 100ml;

1% phosphomolybdic acid: phosphomolybdic acid 1g, distilled water 100ml;

(2) by liquid-solid for tissue slice Bouin fixed, dewaxing, enter water;

(3) section immerses 2.5% iodine tincture 3 minutes, distillation washing 3 times;

(4) section immerses 5% sodium thiosulfate 5 minutes, distillation washing 3 times;

(5) section immerses 1.5% periodic acid 15 minutes, and oxidizing temperature is between 20-25 DEG C, and distillation is washed 3 times.

(6) take silver dye A liquid 30ml to filter and preheat 25 minutes in 70 DEG C;

(7) silver contaminating B liquid 0.82ml add in preheated silver dye A liquid, section is put in mixing, 70 DEG C contaminate 15 minutes after the coloring case of every 5 minutes Microscopic observation basement membrane, terminate after black clearly until basement membrane shows; Distillation washing 3 times;

(8) 3% sodium thiosulfate soak section 15 seconds, wash 3 minutes × 3 times;

(9) haematoxylin is contaminated 15 minutes, washing;

(10) hydrochloride alcohol differentiation, indigo plant is returned in washing;

(11) Ponceaux dye liquor is contaminated 12 minutes, washes 1 minute × 3 times;

(12) 1% phosphomolybdic acid soak section 1 minute, directly enter 1% aniline blue and contaminate 4 minutes;

(13) to enter dimethylbenzene after gradient alcohol dehydration transparent in section, neutral gum mounting.

Further feature according to hexamine Yin Masong resisdye method of the present invention, described distilled water is electronic grade ultrapure water or Shuan Zheng assay laboratory one-level water.

The hexamine Yin Masong resisdye method of improvement of the present invention, the reagent concentration proportionate relationship of dye liquor, dyeing time, dyeing temperature and the proportioning of resisdye dye liquor, step have been carried out improvement and adjusted, find out the allotment of a kind of reagent rationally, Color is good and the time is short, stable performance, resisdye lovely luster method, and achieve effect more better than existing method in an experiment, change Silver stain is organized and dye easily occurred, time, the more difficult control of temperature, the problem such as background color is too high and resisdye other dyeing color is bright-coloured not.

In dyeing course, the reduction of argent is committed step, often determines tinctorial success or failure. Hexamethylenetetramine in amine Silver stain reagent, boric acid, Borax, silver nitrate proportioning suitable, can prevent that the excessive amine black particle contamination tissue of silver affects the observation of lesion tissue. The present invention allocates out the optimum proportioning of hexamethylenetetramine, boric acid, Borax, silver nitrate in amine Silver stain reagent in constantly groping, and relatively the argentation conventional both at home and abroad such as Gomori method, Jone ' s method, arrow island (Jenos) improved method, Gordon-Sweet method, Janes method decreases the content of silver nitrate. In dye liquor, the content of silver ion reduces, and decreases amine Argent grain and pollutes the possibility of tissue. But silver ion reduces the time making the painted needs of basement membrane longer in dye liquor, the reaction of long amine silver easily makes dye liquor become cloudy and the reflective membrane of black occurs, it is difficult to dispose the reflective membrane of section upper black during auric chloride toning, causes section background dirtier, be unfavorable for the judgement of coloration result. Therefore, the present invention, while adjusting proportioning, have adjusted the time of the temperature of dyeing and dyeing, and before making dye liquor not form muddy and reflective membrane, the dyeing of basement membrane is just complete, and does not need the auric chloride toning in conventional silver dye to remove unnecessary Ag-NOR. This proportioning decreases the background coloration of nephridial tissue brownish black, is more conducive to double staining below.

In the process of dyeing, nephridial tissue is after acid oxidase, makes the mucopolysaccharide in basement membrane expose aldehyde radical, and aldehyde radical is reduced to the argent of pitchy hexamine silver and makes the basement membrane of glomerule painted. Tissue oxidizing is the successful premise that dyes. Oxidation fully just must can make aldehyde radical expose completely, if oxidation is complete not, section silver is painted shallower, affects result and judges. The present invention improves the content of periodic acid in the process aoxidized, and extends the response time of periodic acid, fully exposes the aldehyde radical in basement membrane, improves access times and the effect duration of dye liquor.

Silver dye process in except the formula of reagent, reaction temperature and time except, the present invention use vessel should soak with concentrated sulphuric acid in advance and rinse well. In dyeing course, dye liquor can not the impact dyeing extrinsic factor such as contacting metal material (such as tweezers). About the water joining dye liquor, the present invention compared for several water joining reagent that laboratory is conventional, including: Dan Zhengshui, distilled water, assay laboratory's one-level power and water level ultra-pure water of being processed by water treatment system. Experiments show that, using the double; two steaming of electronic grade ultrapure water and assay laboratory's one-level water to join silver transfection reagent background after processing minimum, dyeing quality is best. Silver dye liquor is with suitable experimental water preparation and can effectively prevent silver from contaminating dye by above-mentioned steps, time and temperature operation, and time, the more difficult control of temperature, background color crosses high insoluble problem.

After nephridial tissue silver hexamine staining, usually carry out Masson double staining, its purpose is to the degree etc. at same the position of deposition of immune complex, form and extent of disease and the kidney region fibrosis showing in glomerule in cutting into slices. Owing to conventional silver hexamine staining dyes black by many in tissue addicted to silver site, this can cause when resisdye horse pine is trichroism in glomerule that the immune complex color that develops the color is light, and basement membrane, mesentery, collagen fiber and kytoplasm, erythrocyte color contrast are not bright-coloured. The present invention changes the formula of classical horse pine dyeing, will mix red dye liquor and change into the proportioning dye liquor of Ponceau S and acid fuchsin, thus eliminate mix red in viride nitens. And find out suitable dyeing time, promoted the bright-coloured degree of the erythrocyte in tissue, Cytoplasm and immune complex and improve the contrast of blue collagen fiber in interstitial.

Accompanying drawing explanation

Fig. 1 is the nephridial tissue section statining figure of the hexamine Yin Masong resisdye method of improvement of the present invention.

Fig. 2 is the nephridial tissue section statining figure (this picture provides from Guangzhou Jinyu Medical Checking Centre professor Geng Jian) of the Jones-HE silver staining that Washington, DC University Medical Center Pathology Deparment adopts.

Fig. 3 be Baoji central hospital adopt Gomori-masson argentation nephridial tissue section statining figure (horse is peaceful, Cai Yuan. the renal fibroblast specimen hexamine silver G horse trichroism resisdye method of pine introduces [J] .2012,19 (10): 391).

Fig. 4 is that Pathology Deparment of Guangdong People's Hospital adopts the nephridial tissue section statining figure (Luo Xinlan vowing that argentation is improved on island (Jenos), Cai Xiu, imperial court is outstanding, Deng. the comparison [J] of glomerular basement membrane silver hexamine staining. clinical and experimental pathology magazine, 200520 (4): 493-494.).

Fig. 5 is domestic various big hospital and medical inspection mechanism nephridial tissue silver hexamine staining figure. The medical history data that image credit provides when Hospital of Southern Medical University (i.e. the applicant) is medical in patient, the pathological section of the silver hexamine staining of patient can be found according to the pathological number of patient to former hospital, from the pathological section that patient brings, find out the coloration result of various big hospital. In figure, A, B come from Pathology Deparment of the first Affiliated Hospital of Guangzhou Zhongshan Medical Univ. (patient's pathological number: K10590); C, D, E come from Guangzhou Jinyu Medical Checking Centre's (patient's pathological number: KB1312967, KB1401240); F comes from Pathology Deparment of Children's Hospital, Jiangxi Prov. (patient's pathological number: 43174).

Fig. 6 is the amine silver reaction contrast under the same conditions of different experimental water. According to figure electronic grade ultrapure water and Shuan Zheng assay laboratory one-level water P < 0.05 compared with one-level use water, single steaming water respectively in one factor analysis of variance, figure, statistically significant.

Detailed description of the invention

The present invention here can be better understood by the detailed description of following specific embodiment. It should be explicitly made clear at this point, these specific embodiments are used only as example rather than any type of restriction to the present invention defined in appended claim. Unless additionally especially definition here, all terms all adopt their the widest possible explanation, including the implication of hint from this specification, and the implication that can know of those skilled in the art and/or in the heavy defined implication such as dictionary, paper.

The following is the part term employed herein explanation in this area.

Dewaxing: paraffin section dyeing must slough the paraffin in section with dimethylbenzene, and effect is that dimethylbenzene can dissolve paraffin, so that dyestuff easily enters cell and tissue.

Differentiation: after brazilwood extract dyeing, wash unconjugated dye liquor with water, but must sloughing with 1% hydrochloride alcohol in conjunction with the dyestuff of absorption in too much dyestuff and cytoplasm in nucleus, it is clearly demarcated that guarantee nucleus and cytoplasm dye, and this process is called differentiation.

Return indigo plant: after differentiation, hematoxylin is in red ionic condition in acid condition, is then in blue-colored ionic state in the basic conditions, in blueness. So washing removing acid after differentiation with water and terminating differentiation, being that the nucleus that hematoxylin is caught becomes in blueness with alkalescent water, this process claims oil blackeite or returns indigo plant.

Dehydration: section statining will be cut into slices by low-concentration ethanol excessively to the process that the ethanol of high concentration is excessive after terminating, and its effect is that the moisture in tissue is replaced as ethanol, in order to dimethylbenzene enters histiocyte and carries out transparent.

Transparent: paraffin tissue sections dyeing must process through dimethylbenzene after processed, makes section transparent, natural gum mounting could be used.

Embodiment 1: hexamine Yin Masong resisdye method of the present invention

1. agent prescription:

1.1 silver medal dye formula:

Silver dye A liquid: distilled water 230ml, 5% Borax 45ml, 4% boric acid 4ml, 15% hexamethylenetetramine 60ml. This liquid is put in 4 DEG C of Refrigerator stores, and effect duration is 2 months. Need after 2 months again to prepare.

Silver dye B liquid: distilled water 100ml, 10g silver nitrate. This liquid needs lucifuge in 4 DEG C of Refrigerator stores, and as found, liquid has black precipitate, need to again prepare.

1.2.15% Borax: distilled water 100ml, 5g Borax. This liquid room temperature, keeps in Dark Place.

1.2.215% hexamethylenetetramine: distilled water 100ml, 15g hexamethylenetetramine, this liquid is put in 4 DEG C of Refrigerator stores, occurs that precipitation need to be prepared again.

1.2.34% boric acid: distilled water 100ml, 4g boric acid. This liquid room temperature preservation.

1.2.41.5% periodic acid: distilled water 100ml, 1.5g periodic acid. This liquid is put in 4 DEG C of Refrigerator stores, need to prevent liquid diluting during use, and after contaminating 100 color sections, this liquid need to be prepared again.

1.2.5 Ponceaux dye liquor: Ponceau S 0.7g, acid fuchsin 0.3g, distilled water 100ml, glacial acetic acid 1ml. Room temperature preservation.

1.2.61% aniline blue: aniline blue 1g, glacial acetic acid 1ml, distilled water 100ml. Room temperature preservation.

1.2.71% phosphomolybdic acid: phosphomolybdic acid 1g, distilled water 100ml. Room temperature preservation.

2. step

The liquid-solid fixed tissue slice of 2.1Bouin, dewaxing, enter water. (preheating the 5th step takes silver dye A liquid 30ml and filters and preheat 25 minutes in 70 DEG C).

2.2 sections immerse 2.5% iodine tincture 3 minutes, distillation washing 3 times.

2.3 cut into slices soaks 5 minutes into 5% sodium thiosulfate, distillation washing 3 times.

2.41.5% periodic acid soaks 15 minutes (oxidizing temperature is between 20-25 DEG C), distillation washing 3 times.

2.5 take out preheated silver dye A liquid 30ml(sees 2.1).

Silver is contaminated B liquid 0.82ml by 2.6 to add in preheated silver dye A liquid, and section is put in mixing, 70 DEG C of dip-dye, the coloring case of every 5 minutes Microscopic observation basement membrane after 15 minutes, until basement membrane shows termination after black clearly; Distillation washing 3 times;

2.7 cut into slices soaks 15 seconds into 3% sodium thiosulfate, washes 3 minutes × 3 times.

2.8 haematoxylins are contaminated 15 minutes, washing.

2.9 hydrochloride alcohol differentiation, indigo plant is returned in washing.

2.10 Ponceaux dye liquors are contaminated 12 minutes, wash 1 minute × 3 times.

2.111% phosphomolybdic acid soaks and within 1 minute, directly enters 1% aniline blue dip-dye 4 minutes. Do not wash Direct Dehydration.

It is transparent that 2.12 sections enter dimethylbenzene after gradient alcohol dehydration, neutral gum mounting.

3. points for attention

Please contaminating with vertical dye vat during 3.1 dyeing, desilver dye A, B liquid other liquid outer are all reusable. Dye vat, section must steep acid, distilled water flushing for several times can be standby.

3.2 stained with the irreversibility of color due to silver, to start under mirror Progressive symmetric erythrokeratodermia when dye was to about 15 minutes and observe Color, glomerular basement membrane clearly painted as well. The silver not reproducible use of dye liquor, the dye liquor near effect duration please shorten dyeing time.

3.3 the 7th step 3% sodium thiosulfate times can not be long, otherwise easily falls sheet.

3.4 in above-mentioned experimental procedure, and the distilled water of employing is electronic grade ultrapure water or Shuan Zheng assay laboratory one-level water.

4. result

Basement membrane is black, and collagen fiber are blue, and kytoplasm, muscle fiber, erythrocyte, immune complex are red, karyon indigo plant brown.

Embodiment 2: the comparison of the hexamine Yin Masong resisdye method of improvement of the present invention and domestic and international various big hospital and medical inspection mechanism nephridial tissue silver hexamine staining

Fig. 1 is the nephridial tissue section statining figure of the hexamine Yin Masong resisdye method of improvement of the present invention. It is clear obvious that the position black lines such as the glomerular basement membrane in nephridial tissue, renal tubular basement membrane, basement membrane of blood vessel are sketched the contours by the hexamine Yin Masong resisdye method improved as shown in Figure 1, the clean background of immune complex outside basement membrane and painted bright-coloured, the dyeing of renal tubular cell, in bright gay color.

From Color, this staining is better than Jones-HE silver staining (Fig. 2), Gomori-masson argentation (Fig. 3), vows that argentation (Fig. 4) is improved on island (Jenos); And it is better than the nephridial tissue silver hexamine staining (Fig. 5) that once my institute's (Hospital of Southern Medical University) consultation of doctors, read tablet, the domestic various big hospital of displaying and medical inspection mechanism are done.

Embodiment 3: different experimental waters amine silver reaction contrast under the same conditions

Choose double; two steamings after singly steaming water, distilled water, the assay laboratory's one-level water processed by water treatment system, assay laboratory's one-level water and process water, each 30 parts of samples of electronic grade ultrapure water, it is separately added into silver dye A liquid and the silver dye B liquid of the present invention, carries out amine silver in baking box 70 DEG C and react 1.5 hours. Each sample being placed in 752 type ultraviolet spectrophotometers measure, wavelength is set as 680nm. Take the average of 30 example measurement results, 0 is adjusted with electronic grade ultrapure water silver transfection reagent liquid, comparing result after statistical analysis as shown in Figure 6, it is minimum that visible use electronic grade ultrapure water and Shuan Zheng assay laboratory one-level water join silver transfection reagent light absorption value, turbidity is minimum, is more suitable for nephridial tissue silver hexamine staining.

Claims (2)

1. the hexamine Yin Masong resisdye method improved, it is characterised in that comprise the following steps:

(1) preparation silver transfection reagent, including:

Silver dye A liquid: distilled water 230ml, 5% Borax 45ml, 4% boric acid 4ml, 15% hexamethylenetetramine 60ml;

Silver dye B liquid: distilled water 100ml, 10g silver nitrate;

5% Borax: distilled water 100ml, 5g Borax;

15% hexamethylenetetramine: distilled water 100ml, 15g hexamethylenetetramine;

4% boric acid: distilled water 100ml, 4g boric acid;

1.5% periodic acid: distilled water 100ml, 1.5g periodic acid;

Ponceaux dye liquor: Ponceau S 0.7g, acid fuchsin 0.3g, distilled water 100ml, glacial acetic acid 1ml;

1% aniline blue: aniline blue 1g, glacial acetic acid 1ml, distilled water 100ml;

1% phosphomolybdic acid: phosphomolybdic acid 1g, distilled water 100ml;

(2) by liquid-solid for tissue slice Bouin fixed, dewaxing, enter water;

(3) section immerses 2.5% iodine tincture 3 minutes, distillation washing 3 times;

(4) section immerses 5% sodium thiosulfate 5 minutes, distillation washing 3 times;

(5) section immerses 1.5% periodic acid 15 minutes, and oxidizing temperature is between 20-25 DEG C, and distillation is washed 3 times;

(6) take silver dye A liquid 30ml to filter and preheat 25 minutes in 70 DEG C;

(7) silver contaminating B liquid 0.82ml add in preheated silver dye A liquid, section is put in mixing, 70 DEG C contaminate 15 minutes after the coloring case of every 5 minutes Microscopic observation basement membrane, terminate after black clearly until basement membrane shows; Distillation washing 3 times;

(8) 3% sodium thiosulfate soak section 15 seconds, wash 3 minutes × 3 times;

(9) haematoxylin is contaminated 15 minutes, washing;

(10) hydrochloride alcohol differentiation, indigo plant is returned in washing;

(11) Ponceaux dye liquor is contaminated 12 minutes, washes 1 minute × 3 times;

(12) 1% phosphomolybdic acid soak section 1 minute, directly enter 1% aniline blue and contaminate 4 minutes;

(13) to enter dimethylbenzene after gradient alcohol dehydration transparent in section, neutral gum mounting.

2. hexamine Yin Masong resisdye method according to claim 1, it is characterised in that: described distilled water is electronic grade ultrapure water or Shuan Zheng assay laboratory one-level water.