Dyeing method and dyeing reagent for dyeing pituitary gland of rat

Abstract

The invention relates to the technical field of biological tissue staining, in particular to a staining method and a staining reagent for staining a pituitary gland of a rat, and the staining method adopts a multi-dye synergistic combination of PAS staining, hematoxylin staining, fast green staining and orange G staining to stain a pituitary gland tissue section of the rat. According to the dyeing method disclosed by the invention, the rat pituitary tissue section is dyed through the synergistic combination of multiple dyes, and the core defects of fuzzy details, unclear boundary of suspected cells, light dyeing and low identification degree of traditional dyeing particles are radically solved through the precise synergy and functional complementation of the dyes.

Description

Technical Field

[0001] This invention relates to the field of biological tissue staining technology, specifically to a staining method and staining reagent for staining rat pituitary tissue. It enables high-resolution, high-stability, and multifunctional staining of pituitary tissue from SD rats, allowing for integrated observation of structural partitioning, cell type identification, and functional status assessment. Background Technology

[0002] In the fields of life science research and pharmaceutical development, SD rats have become one of the most widely used experimental animals due to their physiological structure, controllable genetic background, and convenient experimental operation characteristics, which are highly similar to those of humans. From basic physiological mechanism research to preclinical drug safety and efficacy evaluation, SD rats have always played a "bridge" role—simulating the pathological process of human diseases and providing researchers with reproducible and quantifiable experimental data, making them a core experimental model for promoting endocrine disease research and non-clinical drug evaluation.

[0003] The rat endocrine axes (such as the hypothalamic-pituitary-thyroid axis and the hypothalamic-pituitary-gonadal axis) are structurally similar to and functionally homologous to those in humans. The rat pituitary gland, as the body's "endocrine center," possesses a unique cellular composition (the anterior pituitary contains eosinophilic, basophilic, and chromophobe cells; the neurohypophysis lacks hormone synthesis capacity; and the middle portion contains melanocyte-stimulating cells) and hormone secretion function (the anterior pituitary synthesizes seven key hormones; the neurohypophysis stores and releases antidiuretic hormone and oxytocin; and the middle portion secretes melanocyte-stimulating hormone). These characteristics make the rat pituitary a core vehicle for non-clinical drug evaluation (safety and efficacy assessment) and the construction of pharmacodynamic models for endocrine diseases.

[0004] In practical research, whether it is to detect the potential toxicity of drugs to the endocrine system (such as assessing whether drugs interfere with the synthesis of pituitary hormones) or to simulate growth, metabolism, and reproductive diseases to verify the efficacy of drugs (such as judging the improvement effect of drugs on endocrine diseases by observing changes in pituitary cell morphology), it is necessary to clearly present the structural divisions and cellular characteristics of the rat pituitary gland through staining techniques in order to obtain accurate experimental data. This makes rat pituitary staining technology an indispensable basic technical link in life science research and pharmaceutical development. The accuracy and convenience of related technologies directly affect the research efficiency and the reliability of the results.

[0005] Currently, HE staining (hematoxylin-eosin staining) is the most widely used staining technique for observing the structure and identifying cells of the rat pituitary gland. Its core principle is that the acidophilic, basophilic, and chromophobe cells of the anterior pituitary gland have different affinities for hematoxylin (a basic dye that makes acidic substances like the cell nucleus appear blue) and eosin (an acidic dye that makes basic substances like the cytoplasm appear red) due to the differences in the chemical properties of the hormone secretion granules within their cytoplasm. Eosinophilic cells, containing a large number of acidic secretion granules, have a strong affinity for eosin, resulting in a bright red cytoplasm; basophilic cells, containing basic secretion granules, have a strong affinity for hematoxylin, resulting in a blue cytoplasm; and chromophobe cells, with fewer secretion granules, have a weak affinity for both dyes, resulting in a pale or colorless cytoplasm. However, in practice, the staining results deviate significantly from the theoretical predictions, and the distinction between acidophilic, basophilic, and chromophobe cells is often unclear.

[0006] By using HE staining, researchers can preliminarily distinguish the three structures of the rat pituitary gland: the adenohypophysis, the neurohypophysis, and the intermediate part.

[0007] However, existing HE staining techniques have significant limitations in rat pituitary staining: First, the cell differentiation accuracy is insufficient, as HE staining alone cannot further differentiate cells, making it impossible to accurately observe the effects of drugs on specific hormone-secreting cells; second, the staining stability is poor—affected by staining time, temperature, and dye concentration, uneven staining may occur in the same batch of pituitary samples (e.g., basophils in some areas appear light blue instead of dark blue), leading to reduced reproducibility of experimental data; third, it cannot simultaneously display cell structure and hormone expression—HE staining can only present cell morphology and structural regions. If further detection of the type of hormone secreted by cells is required, it must be combined with immunohistochemical staining techniques, increasing experimental steps and costs.

[0008] In summary, while existing rat pituitary staining techniques based on HE staining can meet general structural observation needs, they have significant shortcomings in terms of cell differentiation accuracy, staining stability, and combined functional and structural visualization. They are no longer adequate for the demands of current life science research and pharmaceutical development (such as precise evaluation of drug toxicity to specific pituitary cells) for high-resolution, high-stability, and multifunctional staining techniques. Therefore, there is an urgent need to develop a novel rat pituitary staining method to address these issues. Summary of the Invention

[0009] In order to overcome the dual technical bottlenecks of insufficient traditional special staining effects and limited application of mainstream immunohistochemistry techniques in existing rat pituitary staining methods, this invention proposes a staining method and staining reagent specifically for rat pituitary staining, taking into account the histological characteristics of the rat pituitary gland.

[0010] The present invention adopts the following technical solution:

[0011] Firstly, a staining method for rat pituitary gland involves using a synergistic combination of multiple dyes, including PAS staining, hematoxylin staining, Fast Green staining, and Orange G staining, to stain rat pituitary tissue sections.

[0012] The rat pituitary gland is divided into three parts: the anterior pituitary gland, the posterior pituitary gland, and the intermediate part.

[0013] Anterior pituitary gland: The core area of ​​endocrine function, where all eosinophilic, basophilic, and chromophobe cells are concentrated, directly synthesizing and secreting 7 key hormones;

[0014] Neurohypophysis: It has no hormone synthesis capacity, but only stores and releases antidiuretic hormone (ADH) and oxytocin (OT) synthesized by the hypothalamus, which regulate water metabolism and reproductive-related physiological activities;

[0015] The middle part: located between the anterior pituitary gland and the neurohypophysis, mainly contains melanocytes (MSH cells), which secrete melanocyte-stimulating hormone (MSH) to regulate skin pigmentation.

[0016] The pituitary gland (especially the adenohypophysis) is composed of various functional cells such as eosinophils, basophils, and chromophobes. The hormones secreted by different cells (such as GH, ACTH, PRL, etc.) are directly related to its physiological functions and pathological states.

[0017] Conventional HE staining yields poor results, characterized by red or bluish-red cytoplasm in both eosinophils and basophils, blurred granule details, and indistinct boundaries and pale staining in chromophobe cells, making the three cell types almost indistinguishable. The method of this invention precisely overcomes this deficiency through a multi-color combination strategy of PAS staining + hematoxylin + Fast Green + Orange G: PAS staining specifically displays the mucopolysaccharide components (purple-red) within basophils; Fast Green and Orange G stain the protein components of different types of eosinophils respectively; and hematoxylin clearly marks the cell nucleus. The four staining methods create a multi-dimensional comparison of "nucleus-cytoplasm-specific components," making the morphology, boundaries, and characteristic granules of each cell clearly distinguishable, thus solving the core problem of "low cell identification" in traditional special staining. The specific mechanism of action is as follows:

[0018] PAS staining (periodic acid-Schiff reaction): specifically identifies basophilic cells.

[0019] Utilizing the characteristic that basophils are rich in mucopolysaccharides (such as glycoproteins in hormone secretion granules), the polysaccharides are oxidized with 1% periodic acid to generate aldehyde groups, which are then combined with Schiff's reagent to form purple-red granules, accurately highlighting basophils and filling the gap that traditional HE staining cannot specifically label these cells, thus solving the problem of "difficulty in identifying functional core cells".

[0020] Hematoxylin: Establishing a benchmark for nuclear localization

[0021] As a classic basic dye, it specifically binds to nucleic acids (acidic substances) in the cell nucleus, turning blue-purple and providing clear "structural anchors" for all cells. This avoids counting errors caused by blurred cell boundaries in traditional staining and also helps to determine cell integrity (such as pathological conditions like nuclear condensation and nuclear fragmentation).

[0022] Fast Green: Targeted Labeling of Eosinophils

[0023] For acidic protein components (such as growth hormone and prolactin secretion granules) in eosinophilic cells, the acid dye Fast Green specifically binds to them, producing a bright green color that contrasts sharply with the purplish-red color of PAS staining. This completely solves the problem of "color confusion" between eosinophilic and basophilic cells in traditional HE staining, enabling rapid differentiation between the two types of functional cells.

[0024] Orange-yellow G: Clearly outlines the interstitial and vascular structures.

[0025] This method specifically stains the connective tissue, blood vessel walls, and other interstitial structures in the pituitary gland, producing a bright yellow color that creates a gradient difference from the color of the parenchymal cells (eosinophilic and basophilic cells) while clearly displaying the morphology of the blood vessel lumen and endothelial cells (which appear dark red). This overcomes the shortcomings of traditional staining methods, such as poor identification of blood vessel structures and mutual obscuring between interstitial and parenchymal cells.

[0026] Moreover, the method of the present invention does not require expensive antibodies and only uses conventional chemical reagents; the process is simplified and can be completed in a few hours; and it can achieve simultaneous color development of all cells on the same slice, which greatly reduces experimental costs and technical barriers, and solves the problems of high cost and complicated operation that exist in the current method of distinguishing pituitary cells by immunohistochemistry.

[0027] Preferably, the above method specifically includes the following steps:

[0028] (a) Prepare paraffin sections and dewax the paraffin sections to water;

[0029] (b) Stain the dewaxed sections with PAS and rinse with running water;

[0030] (c) After PAS staining, the sections were stained with hematoxylin and rinsed with running water;

[0031] (d) After hematoxylin staining, the sections were stained with fast green and rinsed with running water;

[0032] (e) After Fast Green staining, the sections were stained with Orange G and rinsed with running water;

[0033] (f) Dehydrate, clear and mount to obtain stainable sections.

[0034] Preferably, the PAS staining step includes:

[0035] (b-1) Incubate the sections with 1% periodic acid solution at 18-22℃ for 5-15 minutes;

[0036] (b-2) Rinse slowly with running water, then soak in distilled water;

[0037] (b-3) Add Schiff's reagent and incubate in the dark for 15-30 minutes;

[0038] (b-4) Rinse with running water and soak in distilled water.

[0039] Preferably, the Schiff reagent is preheated to room temperature before use.

[0040] Preferably, the hematoxylin staining time in step (c) is 3-8 minutes.

[0041] As a preferred method, after hematoxylin staining, differentiation is performed using 1% hydrochloric acid ethanol for 5-15 seconds.

[0042] Preferably, the Fast Green staining time is 1-3 minutes.

[0043] As a preferred method, the staining time for Orange G is 1-3 minutes.

[0044] Preferably, the slice thickness is 1-3 μm.

[0045] To address the stability issues of traditional HE staining, which is greatly affected by temperature and concentration and results in uneven color development, a standardized reagent preparation process and quality control standards are implemented to ensure consistent staining results. Key technical details include:

[0046] 0.5% Orange G staining solution:

[0047] ① Measure 50 mL of distilled water into a beaker, add 1 g of orange yellow G powder, and stir with a magnetic stirrer for 5-10 minutes until completely dissolved;

[0048] ② Add 2.5g of phosphotungstic acid hydrate and continue stirring for 15 minutes to ensure no particles remain;

[0049] ③ No filtration required. Transfer directly to a brown reagent bottle and store at room temperature away from light. Shelf life is 1 month.

[0050] Strictly control the ratio of Orange Yellow G (1g), phosphotungstic acid hydrate (2.5g) to distilled water (50mL), stir magnetically to ensure no particle residue, store at room temperature away from light (shelf life 1 month), and avoid uneven dyeing caused by dye precipitation.

[0051] 1% periodic acid solution:

[0052] ① Weigh 1g of periodic acid powder and slowly add it to 100mL of distilled water while stirring until dissolved;

[0053] ② Transfer to a brown reagent bottle and store at 4°C protected from light. Allow to reach room temperature 30 minutes before use. Shelf life is 12 months.

[0054] Store at 4℃ away from light (shelf life 12 months). Allow the product to return to room temperature (18-22℃) 30 minutes before use. Strictly control the oxidation temperature to prevent excessive oxidation from causing false negative PAS staining.

[0055] Schiff's reagent:

[0056] ① After boiling 200mL of distilled water, add 1g of basic fuchsin and continue boiling for 5 minutes until completely dissolved;

[0057] ② Filter the solution after cooling it to 50°C, and add 20 mL of 1 mol / L HCl after cooling the filtrate to 25°C;

[0058] ③ Add 1g of potassium metabisulfite, shake well, seal the bottle tightly, wrap it with black paper, and place it in a refrigerator at 4℃ for 24 hours;

[0059] ④ Add 0.5g of activated carbon and shake for 1 minute. After filtration, the solution is colorless and transparent and ready for use. Store at 4℃ away from light for six months.

[0060] ⑤ Failure judgment: If the solution turns pink, it needs to be prepared again.

[0061] By using a process of "boiling and dissolving - gradient cooling - activated carbon decolorization" (such as cooling to 50℃ and filtering, adding HCl at 25℃), the solution is ensured to be colorless and transparent, and the failure judgment criteria are clear (if it turns pink, it needs to be prepared again), thus avoiding the weakening of the staining signal due to reagent failure.

[0062] Secondly, the present invention provides a staining reagent for staining rat pituitary gland, including hematoxylin staining solution;

[0063] 1% periodic acid solution, Schiff's reagent, hematoxylin staining solution, 0.5% orange-yellow G staining solution, and fast green staining solution.

[0064] By implementing the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0065] This invention synergistically combines PAS staining, hematoxylin staining, Fast Green staining, and Orange G staining to stain rat pituitary tissue sections. Through the precise synergy and functional complementarity of each dye, it fundamentally solves the core deficiency of low discrimination in traditional staining: Hematoxylin, as a classic nuclear staining agent, specifically binds to nucleic acids within the cell nucleus, producing a blue color, thus establishing a clear "structural benchmark" for the pituitary tissue and making the location of the cell nucleus clearly identifiable; PAS (periodic acid-Schiff reaction) reagent can accurately identify polysaccharides in the pituitary (such as glycoproteins in basophil secretory granules), forming a purple-red staining signal. This characteristic fills the gap in traditional staining's inability to clearly distinguish basophils from other cell types, enabling… Specific cell populations with secretory functions are highlighted; Fast Green targets acidic protein components in the cytoplasm, combining with acidic substances in eosinophilic cells to appear green, forming a sharp color contrast with the purplish-red of PAS, further refining the distinguishing dimensions of cytoplasmic components and avoiding the "mixing of colors" of different cytoplasmic components in traditional single staining; Orange-Yellow, as a supplementary staining agent, can specifically stain the connective tissue, blood vessel walls and other interstitial structures in the pituitary gland, presenting a bright orange-yellow color, which not only forms a multi-color gradient difference with the blue of the cell nucleus, the green of eosinophilic cells and the purplish-red of basophilic cells, but also clearly outlines the overall structure of the pituitary tissue, solving the defect of mutual obscuring between interstitial tissue and parenchymal cells in traditional staining. From a practical application perspective, this combination offers several advantages. It requires no complex or specialized equipment and can be achieved through conventional staining steps, making it convenient to use. All dyes used are mature, conventional staining agents, eliminating the need for additional research and development or procurement of special reagents, thus reducing experimental costs and meeting the demand for low-cost solutions. Simultaneously, the synergistic staining effect of multiple colors significantly enhances the identification of various pituitary components (nucleus, eosinophils, basophils, and interstitial structures), with staining performance far exceeding traditional single or dual-color staining, achieving high-performance standards. This provides precise morphological evidence for pathological diagnosis and histological research, effectively improving the quality of experiments and diagnoses. Attached Figure Description

[0066] Figure 1 The results of staining rat pituitary tissue using the staining method described in Example 1 of this invention;

[0067] Figure 2 The results show the staining of rat pituitary tissue using the staining method of Comparative Example 1.

[0068] Figure 3 The results show the staining of rat pituitary tissue using the staining method in Comparative Example 2.

[0069] Figure 4 The results show the staining of rat pituitary tissue using the staining method described in Comparative Example 3. Detailed Implementation

[0070] The technical solution of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0071] Unless otherwise specified, the raw materials used in the following embodiments are all conventional materials in the art.

[0072] Example 1: Multi-dye synergistic staining of pituitary tissue from normal SD rats

[0073] 1. Materials and Reagents

[0074] Experimental animals: Healthy adult male SD rats, weighing 200–250g.

[0075] Tissue sampling: Pituitary tissue was quickly removed after euthanasia and fixed in 4% buffered neutral formalin for 24 hours.

[0076] Embedding and sectioning: conventional gradient alcohol dehydration, xylene clearing, paraffin embedding, and continuous sectioning with a fully automated microtome to a thickness of 2μm.

[0077] Reagents:

[0078] (a) Preparation method of self-prepared reagents:

[0079] 0.5% Orange G staining solution (Aladdin reagent)

[0080] Ingredients: 1g of Orange Yellow G (O100203), 2.5g of phosphotungstic acid hydrate (P100467), 50mL of distilled water

[0081] step:

[0082] ① Measure 50 mL of distilled water into a beaker, add 1 g of orange yellow G powder, and stir with a magnetic stirrer for 5-10 minutes until completely dissolved;

[0083] ② Add 2.5g of phosphotungstic acid hydrate and continue stirring for 15 minutes to ensure no particles remain;

[0084] ③ No filtration required. Transfer directly to a brown reagent bottle and store at room temperature away from light. Shelf life is 1 month.

[0085] 1% periodic acid solution

[0086] Ingredients: 1g periodic acid (analytical grade), 100mL distilled water

[0087] step:

[0088] ① Weigh 1g of periodic acid powder and slowly add it to 100mL of distilled water while stirring until dissolved;

[0089] ② Transfer to a brown reagent bottle and store at 4°C protected from light. Allow to reach room temperature 30 minutes before use. Shelf life is 12 months.

[0090] ③Note: The oxidation temperature should be controlled at 18-22℃ to avoid excessive oxidation that could lead to false negative staining.

[0091] Schiff's reagent (aldehyde-specific colorimetric reagent)

[0092] Ingredients: 1g basic fuchsin, 20mL 1mol / L HCl, 1g potassium metabisulfite, 0.5g activated carbon, 200mL distilled water

[0093] step:

[0094] ① After boiling 200mL of distilled water, add 1g of basic fuchsin and continue boiling for 5 minutes until completely dissolved;

[0095] ② Filter the solution after cooling it to 50°C, and add 20 mL of 1 mol / L HCl after cooling the filtrate to 25°C;

[0096] ③ Add 1g of potassium metabisulfite, shake well, seal the bottle tightly, wrap it with black paper, and place it in a refrigerator at 4℃ for 24 hours;

[0097] ④ Add 0.5g of activated carbon and shake for 1 minute. After filtration, the solution is colorless and transparent and ready for use. Store at 4℃ away from light for six months.

[0098] ⑤ Failure judgment: If the solution turns pink, it needs to be prepared again.

[0099] Hematoxylin stain (for nuclear staining)

[0100] Ingredients: 6g hematoxylin, 100mL anhydrous ethanol, 150g potassium aluminum sulfate, 2000mL distilled water, 1.2g sodium iodate, 120mL glacial acetic acid, 900mL glycerin

[0101] step:

[0102] ① Dissolve hematoxylin in anhydrous ethanol for later use; dissolve potassium aluminum sulfate in distilled water until completely dissolved;

[0103] ② Mix the two solutions together, add glycerol and stir well, and finally add glacial acetic acid and sodium iodate in sequence;

[0104] ③ Store at room temperature away from light. Use 7 days after maturation. Shelf life is 6 months.

[0105] (II) Preparation of finished reagents

[0106] Reagent Name Item number supplier Storage conditions 1% Brilliant Green Staining Solution (SF) G1270 Solarborg Room temperature, away from light Fast Green Staining Solution G1371 Solarborg Room temperature, away from light

[0107] 2. Staining steps

[0108] Follow these steps:

[0109] (a) Dewaxing paraffin sections to water

[0110] Soak in xylene I for 15 minutes → Soak in xylene II for 15 minutes → Soak in xylene: anhydrous ethanol (1:1) for 2 minutes;

[0111] 100% ethanol I for 5 min → 100% ethanol II for 5 min → 80% ethanol for 5 min → rinse with distilled water for 5 min.

[0112] (b) PAS staining

[0113] Place in 1% periodic acid solution and incubate at room temperature (18-22℃) for 10 min;

[0114] Rinse slowly with running water for 2 minutes, then rinse once with distilled water.

[0115] Add Schiff's reagent and incubate in the dark for 15 minutes.

[0116] Rinse with running water for 3 minutes, then briefly soak in distilled water.

[0117] (c) Hematoxylin staining

[0118] Immerse in hematoxylin staining solution and stain at room temperature for 5 minutes;

[0119] Rinse with running water for 2 minutes. If the staining is too dark, add 1% hydrochloric acid ethanol for differentiation for 30 seconds, then rinse with water to restore blue color.

[0120] (d) Cytoplasmic and structural staining

[0121] Add the solid green staining solution (G1371) directly and stain for 2 minutes;

[0122] Rinse quickly with distilled water 3 times (2 seconds each time) to remove residual dye solution from the surface;

[0123] Add 0.5% Orange G staining solution and stain for 1 minute;

[0124] Rinse the slices quickly three times with distilled water and then blot dry the surface of the slices.

[0125] (e) Dehydration, clearing and sealing

[0126] Dehydrate by soaking in anhydrous ethanol for 1 minute;

[0127] Xylene I clears for 2 minutes → Xylene II clears for 2 minutes;

[0128] Use neutral resin to seal the film and avoid air bubbles.

[0129] 3. Observation and Interpretation of Staining Results

[0130] See the results observed under an optical microscope. Figure 1 Basophils have purplish-red granular cytoplasm; eosinophils have bright green cytoplasm; chromophobe cells have unstained cytoplasm; all cell nuclei are blue-purple; blood vessel lumens are bright yellow; and endothelial cells are dark red.

[0131] Comparative Example 1:

[0132] HE staining was used to stain tissues from normal SD rats (from the same group as in Example 1). The specific steps are as follows:

[0133] (a) Dewaxing

[0134] The paraffin slices were baked in an oven at 65°C for 5 minutes; dewaxed by xylene I and xylene II for 8 minutes each; soaked in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each; then treated in ethanol solutions of 90%, 80%, and 70% for 3 minutes each; and rinsed with running water for 3 minutes.

[0135] (b) Staining

[0136] Immerse the sections in hematoxylin staining solution for 5 minutes, then rinse with running water for about 3 minutes; immerse the sections in differentiation solution for 8 seconds, then rinse with running water for 3 minutes; immerse the sections in dilute ammonia solution for 1 minute to achieve blue reversion, then rinse with running water for another 2 minutes; immerse the sections in 80% ethanol II and 95% ethanol I for 3 minutes each; perform comparative staining with eosin staining solution for 1 minute; immerse the sections in 95% ethanol II and 95% ethanol III for 80 seconds each, then immerse them in anhydrous ethanol III and anhydrous ethanol IV for 5 minutes each.

[0137] (c) Dehydration, clearing and sealing

[0138] Dehydrate by soaking in anhydrous ethanol for 1 minute; clear with xylene I for 2 minutes → clear with xylene II for 2 minutes; seal with neutral resin to avoid air bubbles.

[0139] See the results observed under an optical microscope. Figure 2 The adenohypophysis, the anterior lobe of the pituitary gland. Glandular cells are arranged in dense, cord-like clusters, with abundant sinusoidal capillaries visible between them. The glandular cells consist of eosinophils, basophils, and chromophobes. Eosinophils have red cytoplasm, are relatively large, and are mostly round or oval in shape. Basophils have purplish-red cytoplasm. Chromophobes are small, with lightly stained cytoplasm. The capillary lumen is homogeneous and red. All types of glandular cells have cytoplasm tinged with red to varying degrees, making them difficult to distinguish.

[0140] Comparative Example 2

[0141] The staining method used in this comparative example is as follows (the difference from Example 1 lies in the different steps for cytoplasmic and structural staining):

[0142] (a) Dewaxing paraffin sections to water

[0143] Soak in xylene I for 15 min → Soak in xylene II for 15 min → Soak in xylene: anhydrous ethanol (1:1) for 2 min; 100% ethanol I for 5 min → 100% ethanol II for 5 min → 80% ethanol for 5 min → Rinse with distilled water for 5 min.

[0144] (b) PAS staining

[0145] Place in 1% periodic acid solution and incubate at room temperature (18-22℃) for 10 min; rinse slowly with running water for 2 min, and rinse once with distilled water; place in Schiff reagent and incubate in the dark for 15 min; rinse with running water for 3 min, and rinse briefly with distilled water.

[0146] (c) Hematoxylin staining

[0147] Immerse in hematoxylin staining solution and stain at room temperature for 5 minutes; rinse with running water for 2 minutes. If the staining is too dark, add 1% hydrochloric acid ethanol for differentiation for 30 seconds and then rinse with water to return to blue.

[0148] (d) Cytoplasmic and structural staining

[0149] Add 1% brilliant green directly and stain for 2 minutes; rinse quickly with distilled water 3 times (2 seconds each time) to remove residual staining solution on the surface; add 0.5% orange yellow G staining solution and stain for 1 minute; rinse quickly with distilled water 3 times and blot dry the surface of the section.

[0150] (e) Dehydration, clearing and sealing

[0151] Dehydrate by soaking in anhydrous ethanol for 1 minute; clear with xylene I for 2 minutes → clear with xylene II for 2 minutes; seal with neutral resin to avoid air bubbles.

[0152] See results Figure 3 , Figure 3 The overall bright green coloration of the tissue is too heavy, affecting the coloration of other parts.

[0153] Comparative Example 3

[0154] The staining method used in this comparative example is as follows (the difference from Example 1 lies in the different steps for cytoplasmic and structural staining):

[0155] (a) Dewaxing paraffin sections to water

[0156] Soak in xylene I for 15 min → Soak in xylene II for 15 min → Soak in xylene: anhydrous ethanol (1:1) for 2 min; 100% ethanol I for 5 min → 100% ethanol II for 5 min → 80% ethanol for 5 min → Rinse with distilled water for 5 min.

[0157] (b) PAS staining

[0158] Place in 1% periodic acid solution and incubate at room temperature (18-22℃) for 10 min; rinse slowly with running water for 2 min, and rinse once with distilled water; place in Schiff reagent and incubate in the dark for 15 min; rinse with running water for 3 min, and rinse briefly with distilled water.

[0159] (c) Hematoxylin staining

[0160] Immerse in hematoxylin staining solution and stain at room temperature for 5 minutes; rinse with running water for 2 minutes. If the staining is too dark, add 1% hydrochloric acid ethanol for differentiation for 30 seconds and then rinse with water to return to blue.

[0161] (d) Cytoplasmic and structural staining

[0162] Add 0.5% Orange G directly and stain for 2 minutes; rinse quickly with distilled water 3 times (2 seconds each time) to remove residual staining solution on the surface; add 1% Brilliant Green and stain for 4 minutes; rinse quickly with distilled water 3 times and blot dry the surface of the section.

[0163] (e) Dehydration, clearing and sealing

[0164] Dehydrate by soaking in anhydrous ethanol for 1 minute; clear with xylene I for 2 minutes → clear with xylene II for 2 minutes; seal with neutral resin to avoid air bubbles.

[0165] See results Figure 4 , Figure 4 The tissue showed bright green but no obvious staining, and the contrast staining was not obvious.

[0166] The foregoing general description of the invention and its specific embodiments should not be construed as a limitation on the technical solution of the invention. Those skilled in the art, based on the disclosure of this application, can add, reduce, or combine the disclosed technical features in the foregoing general description and / or specific embodiments (including examples) without departing from the constituent elements of the invention, to form other technical solutions within the scope of protection of this application.

Claims

1. A staining method for staining a rat pituitary, characterized by, The paraffin sections are dyed by using a multi-dye cooperative combination of PAS staining, hematoxylin staining, fast green staining and orange G staining.

2. The staining method according to claim 1 for staining a rat pituitary, characterized by, Specifically comprising the following steps: (a) preparing paraffin sections and deparaffinating the paraffin sections to water; (b) performing PAS staining on the deparaffinated sections and washing with running water; (c) performing hematoxylin staining on the sections after PAS staining and washing with running water; (d) performing fast green staining on the sections after hematoxylin staining and washing with running water; (e) performing orange G staining on the sections after fast green staining and washing with running water; (f) dehydrating, transparentizing and mounting the sections to obtain dyeable sections.

3. The staining method according to claim 2 for staining a rat pituitary, characterized by, The PAS staining step comprises: (b-1) incubating the sections with 1% potassium iodate solution at a temperature of 18-22°C for 5-15 minutes; (b-2) washing slowly with running water and immersing in distilled water; (b-3) adding Schiff reagent dropwise and incubating in the dark for 15-30 minutes; (b-4) washing with running water and immersing in distilled water.

4. The staining method according to claim 3, wherein The Schiff reagent is preheated to room temperature before use.

5. The staining method according to claim 2, wherein The hematoxylin staining time in step (c) is 3-8 minutes.

6. The staining method according to claim 2, wherein After hematoxylin staining, 1% hydrochloric acid ethanol is used for differentiation for 5-15 seconds.

7. A staining reagent for staining a rat pituitary, characterized by comprising: The hematoxylin staining solution, 1% potassium iodate solution, Schiff reagent, hematoxylin staining solution, 0.5% orange G staining solution and fast green staining solution are included.

8. The staining reagent for staining a rat pituitary according to claim 7, characterized by, The 0.5% orange G staining solution is configured as follows: ① 50mL distilled water is measured in a beaker, 1g orange G powder is added, and a magnetic stirrer is used for stirring for 5-10 minutes until complete dissolution; ② 2.5g phosphotungstic acid hydrate is added, and stirring is continued for 15 minutes to ensure no particle residues; ③ without filtration, it is directly transferred to a brown reagent bottle, and stored at room temperature in the dark, with an effective period of no more than 1 month.

9. The staining reagent for staining a rat pituitary according to claim 7, characterized by, The 1% potassium iodate solution is configured as follows: ① 1g potassium iodate powder is slowly added to 100mL distilled water, and stirring is continued until dissolution; ② it is transferred to a brown reagent bottle, and stored at 4°C in the dark, and before use, it is restored to room temperature in advance, with an effective period of no more than 12 months.

10. The staining reagent for staining of rat pituitary according to claim 7, wherein The Schiff reagent is configured as follows: ① 200mL distilled water is boiled, 1g basic fuchsin is added, and boiling is continued for 5 minutes until complete dissolution; ② when cooled to 50°C, it is filtered, and after the filtrate is cooled to 25°C, 20mL 1mol / L HCl is added; ③ 1g potassium metabisulfite is added, the bottle is tightly plugged after shaking, and it is placed in a 4°C refrigerator for 24 hours; ④ 0.5g activated carbon is added and shaken for 1 minute, and after filtration, the solution is colorless and transparent and can be used, and it is stored at 4°C in the dark.

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