AB-PAS Alsin blue periodate Schiff reagent staining kit and application thereof

Abstract

The invention relates to the technical field of biology, and discloses an AB-PAS (Acetamine-Polyacrylamide) Alpha blue periodate Schiff reagent staining kit, which comprises an Alpha blue acetic acid aqueous solution, a periodate aqueous solution, a Schiff reagent, a sodium hydrogen sulfite solution and a 1N hydrochloric acid solution, the solution is prepared from the following raw materials by mass: 1-2 g of periodate, 1-5 g of Alsin blue, 3-30 ml of glacial acetic acid, 20-30 ml of hydrochloric acid, 1-5 g of basic fuchsin, 1-5 g of sodium hydrogen sulfite, 2 g of activated carbon, 5-20 g of hematoxylin, 1-20 g of potassium alum, 2-20 g of sodium hydrogen sulfite, and 2000 ml of pure water. According to the kit, reagents and chemical components in tissue cells are subjected to specific reaction, or some groups of a dye molecular structure are specifically combined with corresponding groups in tissues to develop color, so that some special components of the tissue cells can be determined; the distribution and the content of the special components in tissue cells and the change of the special components during lesion can be displayed.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more specifically to an AB-PAS alcian blue periodic acid Schiff reagent staining kit and application thereof. Background Art

[0002] Special staining and histochemical staining are staining techniques developed on the basis of HE staining. They are one of the important auxiliary techniques in clinical pathological diagnosis. Many diseases that are difficult to diagnose by conventional HE staining can be further confirmed by applying special staining and histochemical staining techniques. Although immunohistochemical technology is widely used at present, many special staining and histochemical staining cannot be replaced by immunohistochemical technology. The application of special staining and histochemical staining techniques helps to improve the level of clinical pathological diagnosis. Histochemical staining is a staining technique that combines histology, cytology and biochemistry. It uses certain reagents or dyes to specifically react with the chemical components in tissue cells. Certain groups in the reaction or dye molecular structure specifically bind to corresponding groups in the tissue to develop color, which can not only determine certain special components of tissue cells, but also show the distribution and content of these special components in tissue cells and the changes that occur during lesions. With the development of pathology, simple HE staining can no longer meet the needs of clinical pathology for observation of tissue cell morphology. It is hoped that there will be a new staining technology that can solve the problem of observing changes in tissue cell morphology, not only observing the variation of tissue components, but also identifying certain similar substances or structures in tissues or cells seen in HE staining (such as pigments, cytoplasmic vacuoles, etc.) and distinguishing pathogens in lesions.

[0003] Glycogen is mainly stored in the cytoplasm of liver cells and muscle cells. The highest content is in liver cells, cardiomyocytes, and skeletal muscle cells, followed by hair follicles, uterine glands, vaginal epithelium, umbilical cord, etc. There are small amounts of glycogen. Its morphological structure is round particles of varying sizes. It is an important substance for generating energy. Its molecular structure is a polymer of the same type of glucose, that is, it is converted from glucose. When the body needs energy, glycogen is broken down into glucose. Glycogen is easily soluble in water and can be easily broken down into glucose under the action of enzymes. Glucose is even more soluble in water. Therefore, glycogen is easily destroyed after the body dies. Its content The amount of glycogen changes significantly or even disappears completely. Due to the above reasons, when fixing tissue sections with ordinary fixative prepared with water as solvent, not only can the morphology of glycogen not be observed by conventional HE staining, but also it is difficult to observe the morphology and distribution of glycogen with special staining that shows glycogen. Therefore, in order to observe and study the content and distribution of glycogen in tissues, fresh specimens must be selected and fixed in time with a fixative that can preserve and fix glycogen, and then the glycogen can be displayed after special staining. Based on this, the present application proposes an AB-PAS Alcian blue periodic acid Schiff reagent staining kit. Summary of the invention

[0004] The invention aims to provide an AB-PAS alcian blue periodic acid Schiff reagent staining kit and application thereof in view of the deficiencies in the prior art.

[0005] The above technical purpose of the present invention is achieved through the following technical scheme: an AB-PAS alcian blue periodic acid Schiff reagent staining kit, comprising an alcian blue acetic acid aqueous solution, a periodic acid aqueous solution, a Schiff reagent, a sodium bisulfite solution and a 1N hydrochloric acid solution, wherein the above solution is made of raw materials with the following mass components: 1-2g of periodic acid, 1-5g of alcian blue, 3-30ml of glacial acetic acid, 20-30ml of hydrochloric acid, 1-5g of basic fuchsin, 1-5g of sodium bisulfite, 2g of activated carbon, 5-20g of hematoxylin, 1-20g of potassium alum, 2-20g of sodium bisulfite and 2000ml of pure water.

[0006] Preferably, the preparation process of the Alcian blue acetic acid aqueous solution is to dissolve 1g of Alcian blue in 97ml of pure water to obtain solution A, and then add 3ml of glacial acetic acid to solution A.

[0007] Preferably, the preparation process of the periodic acid aqueous solution is to dissolve 1g of periodic acid in 100ml of pure water.

[0008] Preferably, the Schiff reagent preparation process is to heat pure water to a boil, remove the heat, add basic fuchsin, and shake it continuously for 5 minutes to dissolve it, then cool the solution to 50°C, add an equivalent amount of hot hydrochloric acid, cool it to 25°C, add sodium bisulfite and shake it, seal the container and store it in a dark place or in a refrigerator for 12-24 hours, then add 2g of activated carbon, shake it for 1 minute and filter it, store the solution in a refrigerator at 4°C, take it out before use, and use it when the solution rises to room temperature.

[0009] Preferably, the sodium bisulfite solution is prepared by adding 2 g of sodium bisulfite into 180 ml of pure water and then adding 20 ml of equivalent hydrochloric acid.

[0010] Preferably, the 1N hydrochloric acid solution is prepared by adding 8.5 ml of hydrochloric acid with a specific gravity of 1.19 and a content of 37% into 91.5 ml of pure water.

[0011] Preferably, the Alcian blue acetic acid aqueous solution, periodic acid aqueous solution, Schiff's reagent, sodium bisulfite solution and 1N hydrochloric acid solution in the kit are packaged in dropper bottles respectively.

[0012] The method for using the above-mentioned AB-PAS Alcian blue periodic acid Schiff reagent staining kit comprises the following steps: a. Paraffin sections 2-3 μm, dewaxed and washed; b. Wash with pure water for 1-2 minutes; c. Stain with Alcian blue acetic acid aqueous solution for 10-20 minutes; d. Rinse with pure water 3-4 times; e. Oxidation with aqueous periodic acid for 5-10 minutes; f. Rinse with pure water 3-4 times; g. Stain with Schiff's reagent for 10-20 minutes; h. Pour off the dye solution and wash directly with sodium bisulfite solution 3 times, 2 minutes each time; i. Rinse with running water for 5-10 minutes; j. Use hematoxylin solution to stain the nucleus for 2-3 minutes. If the staining is excessive, use 0.5% hydrochloric acid alcohol to differentiate; k. Wash thoroughly to restore blue; l. Dehydrate with alcohol step by step, make it transparent with xylene, and seal it with neutral gum.

[0013] The kit provided by the invention is used in identifying intracellular vacuolar degeneration, diagnosing heart disease and cardiovascular disease, diagnosing glycogen storage disease and diagnosing diabetes.

[0014] Compared with the prior art, the solution of the present invention has the following beneficial effects: The kit of the present invention uses reagents to react specifically with chemical components in tissue cells or certain groups in the dye molecular structure specifically bind to corresponding groups in the tissue to develop color. It can not only determine certain special components of tissue cells, but also show the distribution and content of these special components in tissue cells and the changes that occur during lesions. It is used for the diagnosis and identification of certain tumors: (1) to identify hepatocellular carcinoma and cholangiocarcinoma, that is, the former is positive for glycogen staining and the latter is negative; (2) to identify rhabdomyosarcoma and granular cell myoblastoma, the former is positive for glycogen staining and the latter is negative; (3) Ewing's tumor of bone, sweat gland tumor, rhabdomyosarcoma, chemoreceptor tumor, chordoma, etc., can all be shown as positive when stained with the reagents of the kit of the present invention, and therefore can serve as an important basis for diagnosis. DETAILED DESCRIPTION

[0015] The invention is further described in detail below through specific examples. The following examples are only illustrative and not limiting of the protection scope of the present invention.

[0016] Example 1 An AB-PAS alcian blue periodic acid Schiff reagent staining kit comprises an alcian blue acetic acid aqueous solution, a periodic acid aqueous solution, a Schiff reagent, a sodium bisulfite solution and a 1N hydrochloric acid solution, wherein the solution is prepared from raw materials of the following mass components: 1-2g of periodic acid, 1-5g of alcian blue, 3-30ml of glacial acetic acid, 20-30ml of hydrochloric acid, 1-5g of basic fuchsin, 1-5g of sodium bisulfite, 2g of activated carbon, 5-20g of hematoxylin, 1-20g of potassium alum, 2-20g of sodium bisulfite and 2000ml of pure water.

[0017] In this embodiment, the preparation process of Alcian blue acetic acid aqueous solution is to dissolve 1g Alcian blue in 97ml pure water to obtain solution A, and then add 3ml glacial acetic acid to solution A. The preparation process of periodic acid aqueous solution is to dissolve 1g periodic acid in 100ml pure water. The preparation process of Schiff's reagent is to heat the pure water to boil, remove the heat, add basic fuchsin, and shake it for 5min to dissolve it, then cool the solution to 50°C, add equivalent hot hydrochloric acid, cool to 25°C, add sodium bisulfite and shake, and seal the container and store it in a dark place or refrigerator for 12-24h. , add 2g of activated carbon, shake for 1min and then filter. The solution is stored in a refrigerator at 4℃, taken out before use, and used when the solution reaches room temperature. The preparation process of sodium bisulfite solution is to put 2g of sodium bisulfite into 180ml of pure water, and then add 20ml of equivalent hydrochloric acid. The preparation process of 1N hydrochloric acid solution is to put 8.5ml of hydrochloric acid with a specific gravity of 1.19 and a content of 37% into 91.5ml of pure water. The Alcian blue acetic acid aqueous solution, periodic acid aqueous solution, Schiff reagent, sodium bisulfite solution and 1N hydrochloric acid solution in the kit are respectively packaged in dropper bottles.

[0018] In this embodiment, the fixation method of the kit generally adopts a mixed fixative containing formaldehyde, glacial acetic acid and picric acid prepared with alcohol as a solvent. This type of fixative has a rapid fixation effect, uses alcohol to prevent glycogen from dissolving in water, uses the expansion and softening effects of glacial acetic acid and picric acid to offset the shrinkage and brittleness of the tissue caused by alcohol, and uses the advantages of formaldehyde's strong penetration and uniform fixation of the tissue. The fixation principle is to coagulate the protein combined with glycogen, and the glycogen is protected by the surrounding coagulated protein membrane, rather than coagulating the glycogen.

[0019] Furthermore, the staining results using the reagents of the kit of the present invention are as follows: acidic mucus substances are blue, neutral mucus substances are red, mixed mucus is purple-red, and cell nuclei are light blue.

[0020] Example 2 In this embodiment, an AB-PAS Alcian blue periodic acid Schiff reagent staining kit is used to identify intracellular vacuolar degeneration: in the HE staining of conventional paraffin sections, if vacuoles of varying sizes appear in the cytoplasm, it may be fatty degeneration, vacuoles formed by the dehydration and transparency process of the lipid solvent, or it may be caused by the loss of glycogen during the fixation process with aqueous solution, or it may be simple vesicular degeneration, or even multiple vacuolar changes exist at the same time and are difficult to distinguish. In order to identify their properties, the staining method is used to prove it.

[0021] Example 3 In this embodiment, an AB-PAS Alcian blue periodic acid Schiff reagent staining kit is used for the diagnosis and research of heart disease and cardiovascular disease. Using a special staining method that displays glycogen, it can be observed that the glycogen in the acute early myocardial necrosis or infarction lesions caused by ischemia and hypoxia is significantly reduced or even disappears, presenting a homogeneous dense staining state, while reactive abnormal glycogen increases around the lesions. Glycogen particles may be infiltrated in the myocardial fibers around the healed myocardial infarction lesions. Glycogen staining can prove that there is a large amount of abnormal glycogen in the myocardial cells of myocardial glycogen storage disease, and a large amount of glycogen is also present in myocardial rhabdomyomas, so it is also called cardiac nodular glycogen storage disease.

[0022] Example 4 In this embodiment, an AB-PAS Alcian blue periodic acid Schiff reagent staining kit is used for the diagnosis and research of glycogen storage disease: a special staining method for displaying glycogen can be used to observe that when glycogen storage disease is caused by congenital genetic defects, a large amount of glycogen is accumulated in organs such as the heart, liver, kidney, skeletal muscle, and reticulum system.

[0023] Example 5 In this embodiment, an AB-PAS Alcian blue periodic acid Schiff reagent staining kit is used for the diagnosis and research of diabetes: In diabetes, glycogen can be widely deposited in organs such as the liver, kidneys, and myocardium. Glycogen granules can be seen deposited in hepatocytes, renal convoluted tubular epithelial cells, and myocardial fibers. The amount of glycogen deposited is closely parallel to the blood sugar level.

[0024] Example 6 In this embodiment, an AB-PAS Alcian blue periodic acid Schiff reagent staining kit is used for the diagnosis and differentiation of certain tumors: (1) Differentiating hepatocellular carcinoma from cholangiocarcinoma, i.e., the former is positive for glycogen staining and the latter is negative; (2) Differentiating rhabdomyosarcoma from granular cell myoblastoma, i.e., the former is positive for glycogen staining and the latter is negative; (3) Ewing's tumor of bone, sweat gland tumor, rhabdomyosarcoma, chemoreceptor tumor, chordoma, etc., can all be shown as positive by glycogen staining, and this feature can be used as an important basis for diagnosis.

[0025] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to the implementation rules as needed, but they are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An AB-PAS Alcian blue periodic acid Schiff reagent staining kit, characterized in that, The invention comprises an acetic acid aqueous solution of Alcian blue, an aqueous solution of periodic acid, a Schiff reagent, a sodium bisulfite solution and a 1N hydrochloric acid solution, wherein the above-mentioned solution is prepared from raw materials of the following mass components: 1-2g of periodic acid, 1-5g of Alcian blue, 3-30ml of glacial acetic acid, 20-30ml of hydrochloric acid, 1-5g of basic fuchsin, 1-5g of sodium bisulfite, 2g of activated carbon, 5-20g of hematoxylin, 1-20g of potassium alum, 2-20g of sodium bisulfite and 2000ml of pure water.

2. The AB-PAS Alcian blue periodic acid Schiff reagent staining kit according to claim 1, characterized in that: The preparation process of the Alcian blue acetic acid aqueous solution is as follows: 1 g of Alcian blue is dissolved in 97 ml of pure water to obtain solution A, and then 3 ml of glacial acetic acid is added to solution A.

3. An AB-PAS Alcian blue periodic acid Schiff reagent staining kit according to claim 1, characterized in that, The preparation process of the periodic acid aqueous solution is to dissolve 1g of periodic acid in 100ml of pure water.

4. The AB-PAS Alcian blue periodic acid Schiff reagent staining kit according to claim 1, characterized in that: The preparation process of the Schiff reagent is as follows: after pure water is heated to a boil, alkaline fuchsin is added to the solution, and the solution is continuously shaken for 5 minutes to dissolve the solution. The solution is then cooled to 50°C, an equivalent amount of hot hydrochloric acid is added, the solution is cooled to 25°C, sodium bisulfite is added and shaken, the container is sealed and stored in a dark place or in a refrigerator for 12-24 hours, 2 g of activated carbon is added, the solution is shaken for 1 minute and then filtered, and the solution is stored in a refrigerator at 4°C. The solution is taken out before use and used when the solution reaches room temperature.

5. The AB-PAS Alcian blue periodic acid Schiff reagent staining kit according to claim 1, characterized in that: The preparation process of the sodium bisulfite solution is as follows: 2 g of sodium bisulfite is added into 180 ml of pure water, and then 20 ml of equivalent hydrochloric acid is added.

6. The AB-PAS Alcian blue periodic acid Schiff reagent staining kit according to claim 1, characterized in that: The preparation process of the 1N hydrochloric acid solution is to add 8.5 ml of hydrochloric acid with a specific gravity of 1.19 and a content of 37% into 91.5 ml of pure water.

7. The AB-PAS Alcian blue periodic acid Schiff reagent staining kit according to claim 1, characterized in that: The Alcian blue acetic acid aqueous solution, periodic acid aqueous solution, Schiff's reagent, sodium bisulfite solution and 1N hydrochloric acid solution in the kit are packaged in dropper bottles respectively.

8. The method for using the AB-PAS Alcian blue periodic acid Schiff reagent staining kit according to claim 1-7, characterized in that: The following steps are included: a. Paraffin sections 2-3 μm, dewaxed and washed; b. Wash with pure water for 1-2 minutes; c. Stain with Alcian blue acetic acid aqueous solution for 10-20 minutes; d. Rinse with pure water 3-4 times; e. Oxidation with aqueous periodic acid for 5-10 minutes; f. Rinse with pure water 3-4 times; g. Stain with Schiff's reagent for 10-20 minutes; h. Pour off the dye solution and wash directly with sodium bisulfite solution 3 times, 2 minutes each time; i. Rinse with running water for 5-10 minutes; j. Use hematoxylin solution to stain the nucleus for 2-3 minutes. If the staining is excessive, use 0.5% hydrochloric acid alcohol to differentiate; k. Wash thoroughly to restore blue; l. Dehydrate with alcohol step by step, make it transparent with xylene, and seal it with neutral gum.

9. Use of the AB-PAS Alcian blue periodic acid Schiff reagent staining kit as described in claims 1-8 in identifying intracellular vacuolar degeneration, diagnosing heart disease and cardiovascular disease, diagnosing glycogen storage disease and diagnosing diabetes.