Immunohistochemical staining bright field chromogenic reagent and preparation method thereof

Abstract

The invention provides an immunohistochemical staining bright field chromogenic reagent and a preparation method thereof, and the chromogenic reagent is mainly prepared from the following raw materials in parts by mass: 5-8 parts of an antibody, 10-15 parts of a buffer solution, 9-12 parts of a confining liquid, 35-40 parts of an antibody diluent, 5-8 parts of a dye and 4-7 parts of a nano material. According to the color developing reagent, the stability of the color developing reagent is improved by adding the nanometer material into the color developing agent, the color developing reagent has the advantages of being good in color developing effect and improving sensitivity and specificity, and the problem that in the prior art, defects exist in the color developing effect, sensitivity and specificity is solved.

Description

Technical Field

[0001] The invention relates to the application field of color developing reagents, and in particular to an immunohistochemical staining bright field color developing reagent and a preparation method thereof. Background Art

[0002] Since its introduction in the 1940s, immunohistochemistry (IHC) has become an important tool in biomedical research and clinical diagnosis. The basic principle of this technology is to use the highly specific binding between antigens and antibodies to make the color developer labeled with antibodies develop color through chemical reactions, so as to observe and locate antigenic substances in tissue cells under a microscope. These antigens can be proteins, peptides, enzymes, hormones, pathogens and receptors, etc.

[0003] The main advantage of IHC technology is that it can provide the ability to detect antigens at the cellular and subcellular levels, which is crucial for understanding the molecular mechanisms of diseases and developing new treatment strategies. In addition, IHC technology has the advantages of simple operation and intuitive results, making it widely used in pathology, oncology, neuroscience and other fields.

[0004] However, despite the many conveniences brought by IHC technology, there are still some defects in color development effect, sensitivity and specificity. For example, background staining often occurs during the color development process due to nonspecific binding or cross-reaction of antibodies, which interferes with the localization and detection of the target antigen. In addition, commonly used color developers such as DAB (3,3'-diaminobenzidine) are easily decomposed under light, resulting in unstable color development and affecting the repeatability of experimental results. For low-abundance antigens, existing IHC technology may not be able to detect effectively and requires higher sensitivity. If the specificity of the antibody used is not strong enough, false positive or false negative results may result, affecting the accuracy of the experiment. Even under optimized conditions, there may still be problems with nonspecific binding, which will affect the localization and quantitative analysis of the antigen.

[0005] In summary, although immunohistochemistry technology has important applications in biomedical research and clinical diagnosis, the existing technology has certain defects in color development effect, sensitivity and specificity.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The first purpose of the present invention is to provide an immunohistochemical staining bright field colorimetric reagent, which improves its stability by adding nanomaterials into the colorimetric reagent, has the advantages of good colorimetric effect, improved sensitivity and specificity, and solves the problems of defects in colorimetric effect, sensitivity and specificity in the prior art.

[0008] The second purpose of the present invention is to provide a method for preparing the above-mentioned immunohistochemical staining bright field colorimetric reagent. The preparation method has simple operation steps, and the previous and subsequent operation steps are closely connected. The prepared colorimetric reagent has a good color development effect, and is relatively excellent in sensitivity, specificity and stability.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] The invention provides an immunohistochemical staining bright field color developing reagent, which is mainly prepared from the following raw materials: by weight, 5-8 parts of antibody, 10-15 parts of buffer solution, 9-12 parts of blocking solution, 35-40 parts of antibody diluent, 5-8 parts of dye, and 4-7 parts of nano material.

[0011] Preferably, by weight, the antibody is 6-7 parts, the buffer solution is 11-14 parts, the blocking solution is 10-11 parts, the antibody diluent is 36-39 parts, the dye is 6-7 parts, and the nanomaterial is 5-6 parts.

[0012] The dosage of each raw material component needs to be controlled within an appropriate range to ensure stable color development and high accuracy of the final experimental results.

[0013] Preferably, the antibody of the reagent material is composed of a novel enzyme and a specific antigen binding site. Specifically, the antibody of the reagent material is composed of a novel enzyme and a specific antigen binding site, and the composition is as follows:

[0014] Selection of novel enzymes: horseradish peroxidase is selected as a novel enzyme that can catalyze the redox reaction of the substrate to produce a colored product for colorimetric detection, or alkaline phosphatase is selected as a novel enzyme that can catalyze the dephosphorylation of the substrate under alkaline conditions and is also used for colorimetric or fluorescent detection;

[0015] Design of antigen binding sites: Design two regions to work in combination, either in the variable region to concentrate the antigen binding site on the Fab region of the antibody, which contains two heavy chain variable regions and two light chain variable regions, or in the constant region to participate in activating immune cells, and to concentrate on the Fc region, which consists of two heavy chain constant regions and is responsible for binding to Fc receptors on other cells of the immune system and stimulating the complement system immune response;

[0016] Binding method: The enzyme is fixed on the antibody molecule by electrostatic action. Using horseradish peroxidase as an antibody, the staining result is more stable and its staining retention effect is better than immunoglobulin.

[0017] Preferably, the nanomaterial is a modified carbon nanomaterial with added metal nanoparticles.

[0018] Preferably, the preparation method of the modified carbon nanomaterial comprises the following steps: uniformly dispersing the carbon nanomaterial in a solvent, adding a surfactant and metal nanoparticles, stirring and then sintering.

[0019] Preferably, the surfactant is a mixture of fatty acid glyceride and polysorbate, with the mass ratio of the two being 2:1;

[0020] Preferably, the metal nanoparticles are a composite of tungsten nanoparticles and copper nanoparticles, which are compounded in a mass ratio of 5:1.

[0021] By compounding two surfactants to increase the coordination of the structure, the effect is better than using a single type of surfactant. Due to its huge surface area, metal nanoparticles can better modify the structure of nanomaterials and improve their binding degree with dyes. Of course, in order to improve its effect, tungsten nanoparticles and copper nanoparticles are specially selected to be compounded in a certain mass ratio.

[0022] Preferably, the dye comprises: a water-soluble fluorescent dye and a madder aqueous solution with a mass percentage concentration of 30-40wt%;

[0023] Preferably, by weight, the water-soluble fluorescent dye is 10-20 parts and the madder aqueous solution is 3-4 parts.

[0024] The dye selected madder is matched with a water-soluble fluorescent dye, which can improve the color development effect after matching with the substance in the fluorescent dye, and the color development effect is better and more stable. In order to improve the dispersibility of the dye itself, the concentration of the madder aqueous solution itself cannot be too high, and the amount added during the compatibility should not be too large. The madder is mainly used as an auxiliary component and its amount does not need to be too large.

[0025] Nanomaterials use nanoparticles as basic building blocks. The addition of surfactants and linking groups to achieve structural optimization can better combine and disperse dyes with nano-sized particles, so that nano-pigments not only inherit the color brightness and depth of dyes, but also obtain the stability and durability unique to nanomaterials. And the stability of nanomaterials can be enhanced by modifying them with metal nanoparticles.

[0026] Preferably, the buffer solution is sodium dihydrogen phosphate, potassium dihydrogen phosphate and sodium chloride.

[0027] Preferably, the method for preparing the buffer solution comprises the following steps:

[0028] Weigh 0.3-0.4 mass parts of potassium dihydrogen phosphate, 0.5-0.9 mass parts of disodium hydrogen phosphate, and 0.3-0.4 mass parts of sodium chloride, dissolve them in water, mix them thoroughly, and then adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide solution.

[0029] When selecting the components of the buffer solution, the present invention needs to be composed of sodium dihydrogen phosphate, potassium dihydrogen phosphate and sodium chloride, because if sodium chloride is not added, the buffer solution has no osmotic pressure and ionic strength, and the color developing reagent will work under non-physiological osmotic pressure conditions, thereby affecting its performance and stability. Sodium chloride helps control the ionic strength of the solution by providing additional ions, and the change of ionic strength will affect the reaction kinetics and equilibrium state of the color developing reagent, thereby making the color development effect reach the best, so sodium chloride is needed in the buffer solution. The mass fraction of sodium chloride also needs to be controlled within the dosage range, and too large or too small will affect its balance.

[0030] Preferably, the blocking solution is composed of goat serum, Tween-20 and buffer solution, and the preparation method is: take 30-40% of the total volume of the buffer solution, and add 2-3wt% of goat serum and 4-5wt% of Tween-20 to the buffer solution.

[0031] The blocking solution requires the participation of buffer solution, and the amount of buffer solution used is also required. If the amount of buffer solution used is insufficient and cannot contain more buffer components, it will not be able to provide a more stable pH environment, and it will not be conducive to maintaining the protein structure and function of the reagent.

[0032] Preferably, the antibody diluent consists of gelatin, phosphate buffered saline and sodium azide NaN3.

[0033] Preferably, the preparation process of the antibody diluent is:

[0034] Add gelatin to 30-40% of the total volume of the buffer solution, heat to 60°C, and cool to form a gelatin solution after it is completely dissolved, wherein the mass percentage of the gelatin is 0.5-1wt%;

[0035] Phosphate buffered saline is mixed with sodium azide, wherein the mass percentage of sodium azide is 0.02-0.2wt%;

[0036] Mix the dissolved gelatin solution and the buffer solution containing sodium azide evenly.

[0037] Similarly, the preparation of antibody diluent also requires the participation of buffer solution, and the concentration of NaN3 also has requirements, otherwise it will affect the accuracy of the experimental results.

[0038] The present invention also provides a method for preparing an immunohistochemical staining bright field color developing reagent, comprising the following steps:

[0039] Prepare a buffer solution, a blocking solution and an antibody diluent, dissolve the dye and the nanomaterial in the remaining volume of the buffer solution, and adjust the pH value between 7.6 and 8.3 in real time;

[0040] Under sterile conditions, the above-prepared antibody, blocking solution and antibody diluent are mixed to form a mixed solution;

[0041] The mixed solution was filtered through a 0.22 μm filter membrane and then sterilized using high pressure steam sterilization.

[0042] In the specific preparation process, various parameters need to be controlled well, because an inappropriate acid-base environment will lead to incomplete interaction between the dye and the nanomaterial. Therefore, failure to adjust the pH value will cause the conditions of the solution to deviate from the optimal state, thus affecting the experimental results.

[0043] Preferably, the temperature in the high pressure steam sterilization method is 120-125°C.

[0044] Preferably, during the subsequent storage process, the storage temperature is between 0-8° C. and the humidity is between 35-40%.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention selects a new enzyme as an antibody. The new enzyme has high catalytic efficiency and can catalyze the conversion of substrates into products more quickly, thereby accelerating the speed of the color development reaction. This catalytic effect enables the reagent to produce an obvious color development effect in a short time. Therefore, using the new enzyme in the reagent is helpful to improve the color development effect of the reagent. At the same time, the dye and the nanomaterial are dissolved in a buffer solution, and the pH value is accurately adjusted between 7.6 and 8.3. This operation can ensure the uniform dispersion of the dye component in the solution and avoid the aggregation or precipitation of the dye caused by improper pH value. The introduction of the nanomaterial as a carrier of the dye component not only increases the stability of the dye, but also improves the ability of the dye to bind to the antigen, thereby increasing the color development sensitivity and specificity of the reagent. DETAILED DESCRIPTION

[0047] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0048] Example 1

[0049] Step 1: Prepare a buffer solution: weigh 0.3 parts by mass of potassium dihydrogen phosphate, weigh 0.9 parts by mass of disodium hydrogen phosphate; weigh 0.4 parts by mass of sodium chloride, dissolve them in water, mix thoroughly, adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide solution, dilute with water and mix well to obtain a buffer solution.

[0050] Step 2: Prepare blocking solution: Take 40% of the total volume of the buffer solution and add it to a volumetric flask, and add 3wt% of goat serum and 5wt% of Tween-20 to the taken out buffer solution.

[0051] Step 3: Prepare antibody diluent: add 40% of the total volume of buffer solution into a volumetric flask, add sodium azide to phosphate buffered saline to ensure that NaN3 is completely dissolved, and the final measured concentration range of NaN3 is 0.2wt%; under sterile conditions, dissolve a given proportion of gelatin in 40% of the total volume of buffer solution, heat to 60°C while stirring, and cool to room temperature after complete dissolution. The mass percentage of gelatin is 0.5wt%; mix the dissolved gelatin solution with the phosphate buffer containing NaN3 to obtain the final antibody diluent.

[0052] Step 4: Select horseradish peroxidase as a new enzyme, design two regions to work in combination, concentrate the antigen binding site in the variable region on the Fab region of the antibody, which contains two heavy chain variable regions and two light chain variable regions, or participate in activating immune cells in the constant region, and concentrate on the Fc region consisting of two heavy chain constant regions, and is responsible for binding to Fc receptors on other cells of the immune system, and stimulating the complement system immune response, and using electrostatic effects to fix the enzyme on the antibody molecule. Use horseradish peroxidase as an antibody.

[0053] Step 5: Preparation of nanomaterials: Disperse carbon nanomaterials evenly in ethanol, add 2wt% surfactant (fatty acid glyceride and polysorbate in a mass ratio of 2:1) and 1wt% metal nanoparticles (tungsten nanoparticles and copper nanoparticles in a mass ratio of 5:1), mix well and sinter at 400-500°C.

[0054] Step 6: Prepare dye: Mix 10 parts by weight of water-soluble fluorescent dye and 3 parts by weight of 40 wt% madder aqueous solution.

[0055] Step 7: After mixing the dye and nanomaterial, dissolve them in the remaining volume of buffer solution and adjust the pH value to between 7.6 and 8.3. The prepared reagent materials are mixed in the following proportions: 8 parts by weight of antibody; 15 parts by weight of buffer solution; 9 parts by weight of blocking solution; 40 parts by weight of antibody diluent; 5 parts by weight of dye; and 7 parts by weight of nanomaterial.

[0056] Step 8: Under sterile conditions, mix the above-prepared antibodies, blocking solution and antibody diluent to form a mixed solution; filter the mixed solution through a 0.22-micron filter membrane, and then sterilize it using high-pressure steam sterilization; divide the prepared mixed solution of dye and nanomaterial into sterile containers, mark the date and ingredients, and store the reagents at an appropriate temperature according to the reagent storage requirements, avoiding light and humidity.

[0057] Example 2

[0058] Step 1: Prepare a buffer solution: weigh 0.4 mass parts of potassium dihydrogen phosphate, weigh 0.5 mass parts of disodium hydrogen phosphate; weigh 0.3 mass parts of sodium chloride, dissolve them in water, mix thoroughly, adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide solution, dilute and mix with water to obtain a buffer solution.

[0059] Step 2: Prepare blocking solution: Take 30% of the total volume of the buffer solution and add it to a volumetric flask, and add 2 wt % of goat serum and 4 wt % of Tween-20 to the taken out buffer solution.

[0060] Step 3: Prepare antibody diluent: take 30% of the total volume of buffer solution and add it to a volumetric flask, add sodium azide to phosphate buffered saline to ensure that NaN3 is completely dissolved, and the final measured concentration range of NaN3 is 0.02wt%; under sterile conditions, dissolve a given proportion of gelatin in 30% of the total volume of buffer solution, heat to 60°C while stirring, and cool to room temperature after complete dissolution. The mass percentage of gelatin is 1wt%; mix the dissolved gelatin solution with the phosphate buffer containing NaN3 to obtain the final antibody diluent.

[0061] Step 4: Select horseradish peroxidase as a new enzyme, design two regions to work in combination, concentrate the antigen binding site in the variable region on the Fab region of the antibody, which contains two heavy chain variable regions and two light chain variable regions, or participate in activating immune cells in the constant region, and concentrate on the Fc region consisting of two heavy chain constant regions, and is responsible for binding to Fc receptors on other cells of the immune system, and stimulating the complement system immune response, and using electrostatic effects to fix the enzyme on the antibody molecule. Use horseradish peroxidase as an antibody.

[0062] Step 5: Preparation of nanomaterials: Disperse the carbon nanomaterials evenly in ethanol and add surface

[0063] 2wt% of active agent (fatty acid glyceride and polysorbate in a mass ratio of 2:1) and 1wt% of metal nanoparticles (tungsten nanoparticles and copper nanoparticles in a mass ratio of 5:1) are mixed evenly and then sintered at 400-500°C to obtain the product.

[0064] Step 6: Prepare dye: Mix 20 parts by weight of water-soluble fluorescent dye and 4 parts by weight of 30 wt% madder aqueous solution.

[0065] Step 7: After mixing the dye and nanomaterial, dissolve them in the remaining volume of buffer solution and adjust the pH value to between 7.6 and 8.3. The prepared reagent materials are mixed in the following proportions: 5 parts by mass of antibody; 10 parts by mass of buffer solution; 12 parts by mass of blocking solution; 35 parts by mass of antibody diluent; 8 parts by mass of dye; and 4 parts by mass of nanomaterial.

[0066] Step 8: Under sterile conditions, mix the above-prepared antibodies, blocking solution and antibody diluent to form a mixed solution; filter the mixed solution through a 0.22-micron filter membrane, and then sterilize it using high-pressure steam sterilization; divide the prepared mixed solution of dye and nanomaterial into sterile containers, mark the date and ingredients, and store the reagents at an appropriate temperature according to the reagent storage requirements, avoiding light and humidity.

[0067] Example 3

[0068] Step 1: Prepare a buffer solution: weigh 0.35 mass parts of potassium dihydrogen phosphate, weigh 0.7 mass parts of disodium hydrogen phosphate; weigh 0.35 mass parts of sodium chloride, dissolve them in water, mix thoroughly, adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide solution, dilute with water and mix well to obtain a buffer solution.

[0069] Step 2: Prepare blocking solution: add 35% of the total volume of the buffer solution to a volumetric flask, and add 2.5 wt % of goat serum and 4.5 wt % of Tween-20 to the taken out buffer solution.

[0070] Step 3: Prepare antibody diluent: take 35% of the total volume of buffer solution and add it to a volumetric flask, add sodium azide to phosphate buffered saline to ensure that NaN3 is completely dissolved, and the final measured concentration range of NaN3 is 0.1wt%; under sterile conditions, dissolve a given proportion of gelatin in 35% of the total volume of buffer solution, heat to 60°C while stirring, and cool to room temperature after complete dissolution. The mass percentage of gelatin is 0.7wt%; mix the dissolved gelatin solution with the phosphate buffer containing NaN3 to obtain the final antibody diluent.

[0071] Step 4: Select horseradish peroxidase as a new enzyme, design two regions to work in combination, concentrate the antigen binding site in the variable region on the Fab region of the antibody, which contains two heavy chain variable regions and two light chain variable regions, or participate in activating immune cells in the constant region, and concentrate on the Fc region consisting of two heavy chain constant regions, and is responsible for binding to Fc receptors on other cells of the immune system, and stimulating the complement system immune response, and using electrostatic effects to fix the enzyme on the antibody molecule. Use horseradish peroxidase as an antibody.

[0072] Step 5: Preparation of nanomaterials: Disperse carbon nanomaterials evenly in ethanol, add 2wt% surfactant (fatty acid glyceride and polysorbate in a mass ratio of 2:1) and 1wt% metal nanoparticles (tungsten nanoparticles and copper nanoparticles in a mass ratio of 5:1), mix well and sinter at 400-500°C.

[0073] Step 6: Prepare dye: Mix 15 parts by weight of water-soluble fluorescent dye and 3.5 parts by weight of 35 wt% madder aqueous solution.

[0074] Step 7: After mixing the dye and nanomaterial, dissolve them in the remaining volume of buffer solution and adjust the pH value to between 7.6 and 8.3. The prepared reagent materials are mixed in the following proportions: 6 parts by mass of antibody; 14 parts by mass of buffer solution; 10 parts by mass of blocking solution; 39 parts by mass of antibody diluent; 6 parts by mass of dye; and 5 parts by mass of nanomaterial.

[0075] Step 8: Under sterile conditions, mix the above-prepared antibodies, blocking solution and antibody diluent to form a mixed solution; filter the mixed solution through a 0.22-micron filter membrane, and then sterilize it using high-pressure steam sterilization; divide the prepared mixed solution of dye and nanomaterial into sterile containers, mark the date and ingredients, and store the reagents at an appropriate temperature according to the reagent storage requirements, avoiding light and humidity.

[0076] Example 4

[0077] The specific operation steps are consistent with those of Example 3, except that the above-prepared reagent materials are prepared in the following proportions: 7 parts by mass of antibody; 11 parts by mass of buffer solution; 11 parts by mass of blocking solution; 36 parts by mass of antibody diluent; 7 parts by mass of dye; and 6 parts by mass of nanomaterial.

[0078] Example 5

[0079] The specific operation steps are consistent with those in Example 3, except that the nanomaterial is carbon nanomaterial.

[0080] Example 6

[0081] The specific operation steps are the same as those in Example 3, except that: only copper nanoparticles are selected as the metal nanoparticles.

[0082] Example 7

[0083] The specific operation steps are the same as those in Example 3, except that the surfactant is only fatty acid glyceride.

[0084] Example 8

[0085] The specific operation steps are consistent with those in Example 3, except that no sodium chloride is added to the buffer solution.

[0086] Example 9

[0087] The specific operation steps are the same as those in Example 3, except that only water-soluble fluorescent dye is used as the dye.

[0088] Experimental Example 1

[0089] The test data of the color development effect, color development sensitivity and specificity of the reagent products prepared in the above Examples 1-9 are as follows:

[0090] Table 1 Test results

[0091]

[0092] Note: The color development effect, sensitivity and specificity are evaluated using a score of 1-10.

[0093] It can be seen from Table 1 above that only by implementing the solution of the present invention can good results be achieved. If any parameter is not within the scope of the present invention, the final color rendering effect may be affected.

[0094] Finally, it is understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the principles and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. An immunohistochemical staining bright field color reagent, characterized in that: The invention is mainly prepared from the following raw materials: by weight, 5-8 parts of antibody, 10-15 parts of buffer solution, 9-12 parts of blocking solution, 35-40 parts of antibody diluent, 5-8 parts of dye and 4-7 parts of nano material.

2. The immunohistochemical staining bright field color developing reagent according to claim 1, characterized in that: 6-7 parts of antibody, 11-14 parts of buffer solution, 10-11 parts of blocking solution, 36-39 parts of antibody diluent, 6-7 parts of dye, and 5-6 parts of nanomaterial.

3. The immunohistochemical staining bright field color developing reagent according to any one of claims 1 to 2, characterized in that: The nano material is a modified carbon nano material with added metal nano particles.

4. The immunohistochemical staining bright field color developing reagent according to claim 3, characterized in that: The preparation method of the modified carbon nanomaterial comprises the following steps: uniformly dispersing the carbon nanomaterial in a solvent, adding a surfactant and metal nanoparticles, stirring and then sintering.

5. The immunohistochemical staining bright field color developing reagent according to claim 4, characterized in that: The surfactant is a mixture of fatty acid glyceride and polysorbate, with the mass ratio of the two being 2:1; Preferably, the metal nanoparticles are a composite of tungsten nanoparticles and copper nanoparticles, which are compounded in a mass ratio of 5:

1.

6. The immunohistochemical staining bright field color developing reagent according to any one of claims 1 to 2, characterized in that: The buffer solution is sodium dihydrogen phosphate, potassium dihydrogen phosphate and sodium chloride; Preferably, the method for preparing the buffer solution comprises the following steps: Weigh 0.3-0.4 mass parts of potassium dihydrogen phosphate, 0.5-0.9 mass parts of disodium hydrogen phosphate, and 0.3-0.4 mass parts of sodium chloride, dissolve them in water, mix them thoroughly, and then adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide solution.

7. The immunohistochemical staining bright field color developing reagent according to claim 6, characterized in that: The antibody diluent consists of gelatin, phosphate buffered saline and sodium azide; Preferably, the preparation method comprises: Add gelatin to 30-40% of the total volume of the buffer solution, heat to 60°C, and cool to form a gelatin solution after it is completely dissolved, wherein the mass percentage of the gelatin is 0.5-1wt%; Phosphate buffered saline is mixed with sodium azide, wherein the mass percentage of sodium azide is 0.02-0.2wt%; Mix the dissolved gelatin solution and the buffer solution containing sodium azide evenly.

8. The immunohistochemical staining bright field color developing reagent according to claim 6, characterized in that: The blocking solution is composed of sheep serum, Tween-20 and buffer solution, and the preparation method is: taking 30-40% of the total volume of the buffer solution, and adding 2-3wt% of sheep serum and 4-5wt% of Tween-20 into the buffer solution.

9. The immunohistochemical staining bright field color developing reagent according to claim 6, characterized in that: The dye comprises: a water-soluble fluorescent dye and a madder aqueous solution with a mass percentage concentration of 30-40wt%; Preferably, by weight, the water-soluble fluorescent dye is 10-20 parts and the madder aqueous solution is 3-4 parts.

10. The method for preparing the immunohistochemical staining bright field color developing reagent according to any one of claims 1 to 9, characterized in that: The steps include: Prepare a buffer solution, a blocking solution and an antibody diluent, dissolve the dye and the nanomaterial in the remaining volume of the buffer solution, and adjust the pH value between 7.6 and 8.3 in real time; Under sterile conditions, the above-prepared antibody, blocking solution and antibody diluent are mixed to form a mixed solution; The mixed solution was filtered through a 0.22 μm filter membrane and then sterilized using high pressure steam sterilization.

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