A method for preventing the exfoliation of melanin

Abstract

A method for preventing the retinal pigment from being removed from the network, comprising the steps of pre-fixing liquid fixation, solidifying agent wrapping shaping, fixing liquid fixation, embedding agent embedding, melanin decomposition liquid decomposing melanin, pre-decoloring liquid pre-decoloring, decoloring liquid decoloring and rinsing liquid elution. A method for removing melanin is provided to effectively avoid the retinal RPE layer cell from being removed from the network, protein denaturation and nucleic acid degradation. The method mainly adopts the following steps: pre-fixing liquid to maintain the original state of tissue cells, solidifying agent to maintain the integrity of tissue structure, melanin decomposition liquid composed of various extracts to decompose melanin particles, pre-decoloring liquid and decoloring liquid to separate melanin, and rinsing liquid to elute the decomposed melanin on the surface of the tissue, etc. Under the premise of ensuring the integrity of the retinal tissue structure, not damaging the surface protein, nucleic acid and cell structure of the tissue section, effectively decomposing the melanin particles in the tissue section, maintaining the biological activity of various organelles on the section surface and meeting the needs of subsequent various histochemical staining.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to a method for preventing retinal degeneration and melanin reduction, and further relating to a method for effectively avoiding retinal RPE layer cell degeneration, cell damage, and nucleic acid degradation during the melanin removal process. Background Technology

[0002] Melanin is a unique class of biomolecular pigments formed by the oxidative polymerization of polyphenols or indole monomers. It is widely distributed in many fungi, plants, animals, and microorganisms, and is classified according to its origin into synthetic melanin and natural melanin. Natural melanin is further divided into eumelanin, allomelanin, and pheomelanin based on the different chemical precursors used in its biosynthetic pathway. Eumelanin is primarily black or brown in color, and its molecular structure mainly contains nitrogen and no sulfur. It is formed through a series of oxidative polymerization reactions. Allomelanin is predominantly black in color, and its main characteristic is the absence of nitrogen in elemental analysis. It is widely found in plants and some microorganisms. Pheoomelanin contains both nitrogen and sulfur, and its color is a slightly reddish brown.

[0003] Most melanin is a hydrophobic and negatively charged macromolecule. It has poor solubility in aqueous solutions and common organic solvents, making it a very stable deposited pigment. Under a microscope, it usually does not appear black, but rather yellowish-brown or brownish-black. It contains sulfur but no iron, is insoluble in water and organic solvents, but can be dissolved after prolonged exposure to strong alkalis and decolorized by strong oxidizing agents. Melanin is commonly found in the basal cells of the squamous epithelium of the skin, the hair bulbs of hair, the iris, choroid, and ciliary body of the eye, and the substantia nigra of the midbrain. Some tumor cells also contain melanin-like substances.

[0004] Melanocytes produce melanin, which is mainly stored in melanin granules in the cytoplasm. This can easily cause blurring of the cellular structure in tissue sections, interfering with and affecting subsequent chemical staining results such as hematoxylin staining and immunohistochemical staining. In particular, the retinal structure of the eye is relatively fragile, and its RPE layer cells can easily separate from the retina in liquid, causing detachment and affecting subsequent experimental observations. This invention mainly utilizes the steps of pre-fixation solution fixation, solidification agent encapsulation, fixation solution fixation, embedding agent embedding, melanin decomposition solution decomposition, pre-decolorization solution pre-decolorization, decolorization solution decolorization, and rinsing solution elution. Without damaging other proteins and cellular structures in the tissue section, it provides different melanin decomposition methods according to different purposes, turning the melanin in the tissue section from brown to colorless and clearly displaying the cellular structure to meet the needs of various subsequent chemical staining. Summary of the Invention

[0005] The purpose of this invention is to provide a method for removing melanin particles that prevents retinal detachment, minimizes cell damage, achieves high pigment removal efficiency, and prevents the degradation of nucleic acids (including DNA, RNA, cDNA, and other nucleic acids). Specifically, it provides a melanin removal method suitable for various tissue section types (e.g., paraffin sections, frozen sections, semi-thin sections, and ultrathin sections). The method mainly consists of the following steps: fixation with a pre-fixative solution, encapsulation with a solidifying agent, fixation with a fixative solution, embedding with an embedding agent, melanin decomposition with a melanin-decomposing solution, pre-decolorization with a pre-decolorizing solution, decolorization with a decolorizing solution, and elution with a rinsing solution. This method decomposes melanin particles while ensuring the integrity of the retinal structure and without damaging the exposed cells, proteins, and nucleic acids on the tissue surface, maintaining cell structure integrity, preserving protein activity, and preventing nucleic acid degradation, thus satisfying subsequent chemical staining and molecular manipulation requirements.

[0006] To achieve the above objectives, the following technical solutions are mainly adopted.

[0007] Pre-fixation solution is used for fixation. Fresh tissues are immediately immersed in the tissue pre-fixation solution for fixation for more than 24 hours after dissection. The pre-fixation solution consists of 1.0%-37.0% formaldehyde, 0.1%-25.0% glutaraldehyde, 1.0%-10.0% paraformaldehyde, 0.1%-1.0% osmium tetroxide, 0.01%-0.1% potassium permanganate, 0.01%-0.1% potassium dichromate, 5.0%-10.0% methanol, 5.0%-10.0% ethanol, 5.0%-10.0% acetic acid, 0.01%-0.05% mercuric chloride, and 1.0%-2.5% picric acid. One or more of the above components are mixed in proportion to form a solution with pH 7.0.

[0008] The tissue is encapsulated and shaped with a fixing agent, then removed from the pre-fixation solution. The tissue at the target site is then trimmed and smoothed with a scalpel. The size of the trimmed tissue depends on the type of section; semi-thin and ultrathin sections require epoxy resin, and the tissue size is generally 2mm. 3 -5mm 3 The tissue size of OCT-embedded sections and paraffin-embedded sections is 3mm. 3 -10mm 3 The solidifying agent is prepared by preheating and dissolving. After the solidifying agent is slightly cooled, it is added to a 1.5ml EP tube. Before the solidifying agent solidifies, the trimmed tissue is lifted with tweezers and suspended in the solidifying agent. The tissue is cooled on a -20℃ freezing stage. After the solidifying agent solidifies, the embedded tissue is removed and the embedded block is trimmed. The solidifying agent is composed of 10-80 parts of paraffin wax, 0.5-5.0 parts of agarose, 0.5-15 parts of epoxy resin, 0.5-10 parts of collodion, 0.5-10 parts of gelatin, 0.5-1.0 parts of carbon wax, and 25-90 parts of Optimal Cutting Temperature Compound (OCT). One or more of the above components are prepared in proportion to form the solidifying agent.

[0009] The tissue block, after being encapsulated and shaped by the solidifying agent, is immediately immersed in the tissue fixative for at least 24 hours for further fixation. The fixative consists of 1.0%-37.0% formaldehyde, 0.1%-25.0% glutaraldehyde, 1.0%-10.0% paraformaldehyde, 0.1%-1.0% osmium tetroxide, 0.01%-0.1% potassium permanganate, 0.01%-0.1% potassium dichromate, 5.0%-10.0% methanol, 5.0%-10.0% ethanol, 5.0%-10.0% acetic acid, 0.01%-0.05% mercuric chloride, and 1.0%-2.5% picric acid, with one or more of the above components mixed in proportion to a pH of 7.0.

[0010] Tissue embedding: Tissue blocks that have been fixed in a fixative solution for a second time are further embedded using different embedding agents depending on the experimental purpose. The embedding agent consists of 10-80 parts paraffin, 0.5-5.0 parts agarose, 0.5-15 parts epoxy resin, 0.5-10 parts collodion, 0.5-10 parts gelatin, 0.5-1.0 parts carbon wax, and 25-90 parts Optimal Cutting Temperature Compound (OCT). One or more of the above components are prepared in proportion to form a pre-embedding agent.

[0011] Tissue section preparation: Paraffin tissue sections: Place the trimmed embedded blocks in a paraffin microtome and section to a thickness of 5μm. Float the sections on a 40℃ warm water spreader to flatten the tissue. Remove the sections and bake them in a 60℃ oven. After the paraffin wax melts in the oven, remove the sections and store them at room temperature for later use. Frozen tissue sections: Fix the embedding stage on the microtome. First, roughly trim the tissue surface to make it smooth before sectioning. The section thickness is 8-10μm. Place a clean glass slide flat on top of the sectioned tissue and attach the tissue to the slide. Label the slides and store them at -20℃ for later use. Semi-thin and ultrathin sections: Place the embedded tissue blocks in a 60℃ oven for 48 hours to polymerize. Remove the embedded blocks and section them using an ultrathin microtome. The thickness of a semi-thin section is generally around 1.5μm, and the thickness of an ultrathin section is 60-80nm. Use a 150-mesh copper screen to retrieve the sections.

[0012] Pretreatment of tissue sections: Dewaxing paraffin tissue sections to water: immerse sections sequentially in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then wash with distilled water; Fixation of frozen sections to water: air-dry frozen sections at room temperature, bake in a 37°C oven for 10-20 min, fix with methanol for 20 min, and wash three times with PBS (pH 7.4) on a decolorizing shaker for 5 min each time; Semi-thin and ultrathin sections: proceed directly to subsequent steps.

[0013] The melanin decomposition solution decomposes melanin. The melanin decomposition solution is mainly composed of 10-30 parts of Aspergillus or Saccharomyces cerevisiae extract or budding short-stem mold extract, 15-25 parts of vitamins and their derivatives, 5-20 parts of arbutin and its derivatives, 10-15 parts of resveratrol and its derivatives, 3-15 parts of safflower seed oil and its derivatives, 1-10 parts of rhodioloside and its derivatives, 5-15 parts of passion fruit crude extract, 5-15 parts of red ginseng crude extract, and 5-15 parts of kiwi fruit crude extract.

[0014] After mixing the above components in proportion, adjust the pH to 6.0-8.0, fix the tissue sections in the decolorization box, place the decolorization box in the decolorization shaker, add the above melanin decomposition solution, immerse the tissue sections in the melanin decomposition solution, decompose at 25-40℃, decompose at 15-150 rpm in the dark under constant temperature and humidity for 2-48 hours.

[0015] The preferred extract of Aureobasidium pullulans is derived from the fermentation broth of Aureobasidium pullulans strain EP1001, which was deposited on April 19, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40158 and classified as Aureobasidium pullulans. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0016] Preferably, purines or pyrimidines and their derivatives include one or more of adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, uridine phosphate, uridine diphosphate, uridine triphosphate, guanosine phosphate, guanosine diphosphate, guanosine triphosphate, cytidine phosphate, cytidine diphosphate, and cytidine triphosphate, as well as one or more of cyclic adenosine monophosphate, cyclic adenosine diphosphate, cyclic adenosine triphosphate, cyclic uridine phosphate, cyclic uridine diphosphate, cyclic uridine triphosphate, cyclic guanosine phosphate, cyclic guanosine diphosphate, cyclic guanosine triphosphate, cytidine phosphate, cytidine diphosphate, and cytidine triphosphate.

[0017] Preferably, arbutin and its derivatives include one or more of arbutin, β-arbutin, α-arbutin, deoxyarbutin and arbutin glycolipids.

[0018] Preferably, vitamins and their derivatives include one or more of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, folic acid, and pantothenic acid.

[0019] Preferably, resveratrol and its derivatives include one or more of cis-resveratrol, trans-resveratrol, trans-resveratrol-3-O-β glycoside and cis-resveratrol-3-O-β glycoside.

[0020] The pre-decolorizing solution is composed of various cations and anions, especially divalent and trivalent metal ions and non-metallic examples, including but not limited to magnesium chloride, magnesium sulfate, sodium chloride, sodium carbonate, ferric chloride, ferric oxide, ferric sulfide, ferrous sulfate, alkali-resistant penetrant OEP-70, sodium dodecyl sulfonate, etc., and a mixture of one or more of these. The pH value of the pre-decolorizing solution after mixing is 7.0-8.5.

[0021] Preferably, the contents of the main components in the pre-decolorizing solution are as follows: magnesium chloride 0.1g / L-1.0g / L, magnesium sulfate 0.05g / L-0.25g / L, sodium chloride 1.5g / L-3.0g / L, sodium carbonate 0.06g / L-1.36g / L, ferric chloride 0.006g / L-0.02g / L, ferric oxide 0.001g / L-0.1g / L, ferric sulfide 0.25g / L-1.0g / L, ferrous sulfate 5.0g / L-8.0g / L, alkali-resistant penetrant OEP-70 1.5g / L-2.8g / L, and sodium dodecyl sulfonate 0.1g / L-0.5g / L.

[0022] The decolorizing solution includes one or more of the following: potassium hypochlorite, hypochlorous acid, sodium hypochlorite, potassium permanganate, dichromic acid, potassium dichromate, ozone, melatonin, glutathione, coenzyme Q10, hydrogen peroxide, sodium silicate, sodium dodecylbenzene sulfonate, sodium dodecyl sulfonate, alkali-resistant penetrant OEP-70, and 84 disinfectant. The pH value of the decolorizing solution after mixing is 7.0-8.5.

[0023] Preferably, the main components in the decolorizing solution are as follows: potassium hypochlorite 0.05g / L-0.2g / L, hypochlorous acid 0.03g / L-0.18g / L, sodium hypochlorite 0.05g / L-0.2g / L, potassium permanganate 0.001g / L-0.01g / L, dichromic acid 0.002g / L-0.015g / L, potassium dichromate 0.009g / L-0.01g / L, ozone 0.0005g / L-0.001g / L, melatonin 0.21g / L-0.5g / L, glutathione 2.5g / L-5.0g / L, and coenzyme Q10. 2.9g / L-10.0g / L, hydrogen peroxide 0.5ml / L-30.0ml / L, sodium silicate 0.45g / L-2.2g / L, sodium dodecylbenzene sulfonate 0.45g / L-0.59g / L, sodium dodecyl sulfonate 1.8g / L-6.5g / L, alkali-resistant penetrant OEP-70 0.99g / L-1.89g / L, 84 disinfectant 0.5ml / L-5.0ml / L.

[0024] The rinsing solution mainly consists of one or more of the following: penetrant, fluorescent whitening agent, oxalic acid, sodium bisulfite, sodium thiosulfate, or sodium thiosulfate. The pH value of the mixed rinsing solution is 6.5-7.9.

[0025] Preferably, the main components in the rinsing solution are: alkali-resistant penetrant OEP-70 1.5g / L-2.8g / L, fluorescent whitening agent 3.5g / L-4.8g / L, oxalic acid 2.5g / L-2.9g / L, sodium bisulfite 0.95g / L-5.0g / L, sodium hyposulfite 0.5g / L-3.0g / L, or sodium thiosulfate 2.5g / L-5.0g / L. Attached Figure Description

[0026] Figure 1 The results of hematoxylin-eosin chemical staining of paraffin sections of mouse eyeballs are shown. The left side is the staining result before demelanization, and the right side is the staining result after demelanization. It can be seen with the naked eye that the melanin granules in the right side image have almost disappeared.

[0027] Figure 2 Immunohistochemical staining results of VEGF protein in paraffin sections of mouse eyeballs. The left side shows the immunohistochemical results before demelanization, and the right side shows the immunohistochemical results after demelanization. It can be seen with the naked eye that the brownish-yellow color on the right side has basically disappeared, indicating that the brownish-yellow color on the left side is a false positive result produced by the melanin reaction.

[0028] Figure 3 Comet gel electrophoresis was used to detect intracellular DNA damage. The top left shows the technical solution of this invention, the top right shows an EDTA-hydrogen peroxide composite solution with a pH of 9.0 (hydrogen peroxide content of 0.75%), the bottom left shows a trichloroisocyanuric acid solution with a pH of 6.5 (trichloroisocyanuric acid concentration of 0.25wt%), and the bottom right shows a 0.25% potassium permanganate solution.

[0029] Figure 4 Flow cytometry was used to detect cell apoptosis. The top left shows the technical solution of this invention, the top right shows an EDTA-hydrogen peroxide composite solution with a pH of 9.0 (hydrogen peroxide content of 0.75%), the bottom left shows a trichloroisocyanuric acid solution with a pH of 6.5 (trichloroisocyanuric acid concentration of 0.25wt%), and the bottom right shows a 0.25% potassium permanganate solution.

[0030] Figure 5 Transmission electron microscopy was used to observe the removal of melanin in ultrathin sections of mice. The top left shows the technical solution of this invention, the top right shows an EDTA-hydrogen peroxide composite solution with a pH of 9.0 (hydrogen peroxide content of 0.75%), the bottom left shows a trichloroisocyanuric acid solution with a pH of 6.5 (trichloroisocyanuric acid concentration of 0.25wt%), and the bottom right shows a 0.25% potassium permanganate solution.

[0031] Figure 6 The integrity of the mouse retinal structure and the removal of melanin were observed by semi-thin sections. The upper left shows the technical solution of this invention, the upper right shows an EDTA-hydrogen peroxide composite solution with a pH of 9.0 (hydrogen peroxide content of 0.75%), the lower left shows a trichloroisocyanuric acid solution with a pH of 6.5 (trichloroisocyanuric acid concentration of 0.25wt%), and the lower right shows a 0.25% potassium permanganate solution. Detailed Implementation Example 1

[0032] Eye melanocytes include the pigment epithelium of the eyeball, the epithelium of the sclera (cornea), the melanocytes in the brown layer of the sclera and the stroma of the uvea, as well as the melanocytes of the ocular adnexa (eyelids and conjunctiva). Therefore, the hematoxylin-eosin chemical staining effect of mouse ocular pathological tissue sections is the most appropriate way to confirm the decomposition effect of the melanin granules in the technical solution.

[0033] Fresh, intact ocular tissue was immediately immersed in 4.0% paraformaldehyde fixative at pH 7.5 for at least 24 hours after dissection, with the volume of the fixative being 10 times the volume of the tissue.

[0034] The tissue was removed from the pre-fixation solution, and the tissue at the target site was trimmed and smoothed with a scalpel. The trimmed tissue was 5mm in size. 3 -9mm 3 ; Preheat and dissolve to prepare 1.0% agarose solidifying agent. After slightly cooling the 1.0% agarose, add it to a 1.5ml EP tube. Before the 1.0% agarose solidifies, use tweezers to pick up the trimmed eyeball tissue and suspend it in the 1.0% agarose. Cool on a -20℃ freezing stage. After solidification, remove and trim the embedded block.

[0035] Melted wax is placed into an embedding frame. Before the wax solidifies, the tissue encapsulated with the solidifying agent is placed into the embedding frame and labeled accordingly. The mixture is then cooled on a -20°C freezing stage. After the wax solidifies, the wax block is removed from the embedding frame and trimmed.

[0036] Place the prepared wax block from the above steps into a paraffin microtome and slice it to a thickness of 5 μm. Float the slices on a 40°C warm water spreader to flatten the tissue. Remove the slices from the slide and bake them in a 60°C oven until the wax melts and the slides are dry. Remove them and store them at room temperature for later use.

[0037] The sections obtained in the above steps were sequentially immersed in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then washed with distilled water.

[0038] The tissue sections obtained in the above steps were fixed in a desaturation box, which was placed in a desaturation shaker. The box was immersed in a melanin decomposition solution containing 25 parts of *Bacillus buddingus* extract, 25 parts of vitamin C, 15 parts of arbutin, 3 parts of resveratrol, 7 parts of safflower seed oil, 7 parts of rhodioloside, 5 parts of passion fruit crude extract, 8 parts of red ginseng crude extract, and 5 parts of kiwi fruit crude extract. The decomposition temperature was 28℃, the desaturation shaker speed was 100 rpm / hr, and the decomposition was carried out in the dark for 48 hours under constant temperature and humidity.

[0039] After the tissue sections were decomposed in the dark for 48 hours in the above steps, the decomposition solution was discarded. The sections were then soaked 1-2 times in a pre-decolorizing solution containing 5.0 g / L ferrous sulfate, 1.5 g / L alkali-resistant penetrant OEP-70, and 0.1 g / L dodecyl sulfonic acid at pH 7.0, for 5 minutes each time. The decolorizing box containing the tissue sections, which had been enzymatically digested, was then immersed in the pre-decolorizing solution. The pre-decolorization temperature was 37°C, the pre-decolorization shaker speed was 80 rpm / hr, and the pre-decolorization was carried out in the dark at constant temperature and humidity for 18 hours.

[0040] For the tissue sections that have been pre-decolorized for 18 hours in the above steps, discard the pre-decolorization solution and soak them 1-2 times in a decolorization solution with a pH of 8.0 containing 30 ml / L 27.5% hydrogen peroxide, 2.0 g / L sodium silicate, 6.0 g / L sodium dodecyl sulfonate, and 1.5 g / L alkali-resistant penetrant OEP-70, for 5 minutes each time. Then, immerse the tissue sections in the decolorization solution for decolorization treatment at a decolorization temperature of 40℃ and a decolorization shaker speed of 100 rpm / hr in the dark for 30 minutes under constant temperature and humidity.

[0041] For tissue sections decolorized for 30 minutes in the above steps, discard the decolorizing solution and soak them 2-3 times in a rinsing solution with a pH of 7.0 containing 5.0 g / L alkali-resistant penetrant OEP-70, 0.3 g / L fluorescent whitening agent, 3.0 g / L oxalic acid, and 4.0 g / L sodium bisulfite, for 10 minutes each time. Then rinse with the rinsing solution at a temperature of 35°C and a shaking speed of 80 rpm / hr, maintaining a constant temperature and humidity for 2-3 times, for 15 minutes each time.

[0042] After destaining, tissue sections were stained with hematoxylin for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue solution, and rinsed with running water. Sections were then dehydrated in 95% alcohol for 1 minute and stained with eosin for 15 seconds. The sections were then sequentially stained with anhydrous ethanol I for 2 minutes, anhydrous ethanol II for 2 minutes, anhydrous ethanol III for 2 minutes, n-butanol I for 2 minutes, n-butanol II for 2 minutes, xylene I for 2 minutes, and xylene II for 2 minutes. After clearing, the sections were mounted with neutral resin and observed under a microscope. The cell nuclei appeared blue, and the cytoplasm appeared red. Figure 1 ).

[0043] Beneficial effect: the melanin granules treated by the method of this invention almost disappear. Figure 1 (Right side) The structure of the eye's tissue cells is clear, and the complete cell structures in the retina, optic nerve, and iris can be clearly seen. Example 2

[0044] Immunohistochemical staining is based on the reaction between antigens and antibodies. The activity and content of antigens in tissues are key factors affecting the immunohistochemical effect. During the removal of melanin granules, strong acids, strong bases and strong oxidants can destroy the exposed protein structure in tissue sections and change the physicochemical properties of proteins, and even cause protein denaturation and inactivation, affecting the binding effect of exogenous antibodies and target proteins in subsequent immunohistochemical staining.

[0045] Based on paraffin sections of mouse eyeball tissue, a gentle melanin destaining process was used to decompose melanin granules in the tissue, reducing interference from melanin on immunohistochemical positive signals and decreasing the occurrence of false positives. The experimental procedures were the same as in Example 1. After rinsing, the slides underwent antigen retrieval, endogenous peroxidase blocking, 3% BSA serum blocking, VEGF protein primary antibody incubation, HRP-labeled goat anti-mouse secondary antibody incubation, DAB staining, hematoxylin counterstaining of cell nuclei, dehydration and mounting, and were then interpreted under a white light microscope. Hematoxylin staining of cell nuclei appeared blue, and DAB-induced positive expression appeared brownish-yellow. Figure 2 ).

[0046] Beneficial effect: After immunohistochemical treatment, melanin generally appears brownish-yellow, and the positive result of immunohistochemical antibody binding to antigen also shows brownish-yellow. Therefore, melanin can interfere with the positive results of immunohistochemical staining of tissue sections. In this example, after melanin decolorization treatment, the tissue sections stained with VEGF antibody showed almost no brownish-yellow positive results (…). Figure 2 (Right side), while tissue sections that have not undergone melanin depigmentation showed a deeper brownish-yellow result after VEGF antibody immunohistochemical staining ( Figure 2 (Left side) This indicates that the brownish-yellow color on the left is a false positive result produced by the melanin reaction. Therefore, demelanin treatment of tissue sections can effectively reduce the interference of false positive signals.

[0047] Example 3.

[0048] The comet assay, also known as single-cell gel electrophoresis, can effectively detect and quantify the degree of DNA single- and double-strand breaks in cells. When various endogenous and exogenous DNA damage factors induce DNA strand breaks in cells, their supercoiled structure is disrupted. Under the action of cell lysis buffer, membrane structures such as the cell membrane and nuclear membrane are damaged, and intracellular proteins, RNA, and other components diffuse into the electrolyte. However, nuclear DNA, due to its large molecular weight, cannot enter the gel and remains in situ. To better demonstrate the technical advantages of this application, mouse eyeball tissue was used as a basis to compare the effects of different melanin treatment methods on nucleic acid (DNA, RNA, cDNA, etc.) damage in tissue sections. The preliminary experimental steps were the same as in Example 1. After rinsing with the washing solution, the tissue was digested with 2.5% trypsin for 2 hours. The digested cells were then subjected to a comet gel electrophoresis experiment. The specific experimental steps were performed according to the instructions of the comet electrophoresis kit (catalog number: C2041M, brand: Beyotime). The specific steps are as follows.

[0049] Preparation of the first gel layer: 180 μl of 1% normal melting point agarose NMA (dissolved in Ca2+- and Mg2+-free phosphate buffer PBS) preheated to 45 ℃ was dropped onto the frosted surface of a preheated glass slide, and a clean coverslip was quickly placed on top. The slide was then placed at 4 ℃ for 10 min to allow it to solidify.

[0050] Preparation of the second gel layer: Take 100 μl of cell suspension and 150 μl of 0.8% low melting point agarose LMA (dissolved in Ca2+- and Mg2+-free phosphate buffer PBS) at 37 ℃ and mix well. Then gently remove the coverslip and drop the LMA containing cells onto the first gel layer. Immediately cover with a clean coverslip and cure at 4 ℃ for 20 min.

[0051] Preparation of the third layer of adhesive: Remove the glass slide, and rapidly add 100 μL of 0.5% low-melting-point agarose onto the second layer of agarose at 37 ℃. Cover with a glass slide and cure at 4 ℃ for 20 min. 3. Lithography: Remove the glass slide and immerse the slide in freshly prepared 4 ℃ lysis buffer for 2 h.

[0052] Unwinding: Remove the slide from the lysis buffer, wash away excess salt in distilled water, and air dry. Pour freshly prepared electrophoresis buffer into the electrophoresis tank, cover the slide with 0.25 cm, cover, and incubate in the electrophoresis buffer for 30 min.

[0053] Electrophoresis: At room temperature, adjust the buffer level to 300 mA and set to 25V for 15 min for electrophoresis.

[0054] Washing and staining: After electrophoresis, place the slide in a petri dish and wash with neutralization buffer for 30 min. After neutralization, stain the slide with 50 μL-100 μL acridine orange (EB) solution for 10 min and rinse with distilled water at least twice.

[0055] Results observation: After staining, examine the sample under a fluorescence microscope as soon as possible. If the staining time is too long, the fluorescence will fade. All of the above processes should be performed in the dark to avoid additional DNA damage.

[0056] The processing group adopts the technical solution of the present invention ( Figure 3 (Top left) Control group 1 used an EDTA-hydrogen peroxide complex solution with a pH of 9.0 (hydrogen peroxide content 0.75%). Figure 3 (Top right), Control group 2 used a trichloroisocyanuric acid solution with a pH of 6.5 (the concentration of trichloroisocyanuric acid was 0.25 wt%). Figure 3 (Lower left)), Control group 3 used 0.25% potassium permanganate solution ( Figure 3 (bottom right) After decolorization with melanin, the four groups were used to prepare single-cell suspensions for comet gel electrophoresis experiments.

[0057] Image analysis of the comet images was performed using Comet Assay Software Pect (CASP 1.2.3 beta 1). Based on the DNA content (tDNA%) in the comet tail cells, the degree of cellular DNA damage was classified into five levels: Level 0: No damage (normal cells) and cell damage rate < 5%; Level 1: Low damage, 5%–20%; Level 2: Moderate damage, 20%–40%; Level 3: High damage, 40%–90%; Level 4: Severe damage, > 95%. The statistical results are as follows:

[0058] Table 1. Statistical table of DNA damage degree detected by comet gel electrophoresis under different melanin removal conditions.

[0059]

[0060] TailDNA% is the percentage of tailed DNA to total DNA. Three independent cells were selected from each group for statistical analysis. AV is the average value of TailDNA% from the three cells in each group, and SD represents the standard deviation.

[0061] Ultimately, it was found that the technical solution adopted in this invention, i.e., the degree of DNA damage in the treatment group, was grade 1 (Table 1, ...). Figure 3 (Top left), the DNA damage level of the other three groups was grade 3 (Table 1, Figure 3 Top right Figure 3 Bottom left Figure 3 (bottom right), from which it can be concluded that the technical solution of the present invention is a method that minimizes damage to nucleic acids (DNA or RNA, etc.).

[0062] Example 4

[0063] To further verify the effect of the technical effect of the present invention on cell structure, mouse eyeball tissue was used as a basis to compare the effects of different melanin treatment methods on cell damage. The preliminary experimental steps were the same as in Example 1, and the treatment group adopted the technical solution of the present invention. Figure 4 (Top left) Control group 1 used an EDTA-hydrogen peroxide complex solution with a pH of 9.0 (hydrogen peroxide content 0.75%). Figure 4 (Top right), Control group 2 used a trichloroisocyanuric acid solution with a pH of 6.5 (the concentration of trichloroisocyanuric acid was 0.25 wt%). Figure 4 (Lower left)), Control group 3 used 0.25% potassium permanganate solution ( Figure 4 (Lower right) The tissue, after being depigmented by melanin and rinsed with rinsing solution, was digested with 2.5% trypsin for 2 hours. The digested cells were then analyzed for apoptosis using flow cytometry.

[0064] Table 2. Statistical table of cell apoptosis detected by flow cytometry.

[0065] Grouping percentage of late apoptosis (%) Early apoptosis percentage (%) Total percentage of apoptosis (%) Processing group 9.05 1.1 10.15 Control group 1 16.13 1.64 17.77 Control group 2 17.96 4.92 22.88 Control group 3 25.18 0.99 26.17 .

[0066] The results show that the treatment group, i.e., the technical solution of the present invention (Table 2), Figure 4 The cell apoptosis rate (top left) was the lowest, and cell damage was minimal, thus better protecting the integrity of cell structures in the tissue section.

[0067] Example 5

[0068] Ultrathin sections are a primary technique for observing subcellular structures in tissues, providing a more direct view of melanin deposition within cells. To further verify the technical effectiveness of this invention, ultrathin sections of mouse tissue were used as a basis to compare the effects of different melanin treatment methods on melanin removal in the ultrathin sections.

[0069] Fresh, intact ocular tissue was immediately immersed in 2.5% glutaraldehyde fixative at pH 7.5 for at least 24 hours after dissection, with the volume of the fixative being 10 times the volume of the tissue.

[0070] The tissue was removed from the pre-fixation solution, and the tissue at the target site was trimmed and smoothed with a scalpel. The trimmed tissue was 3mm in size. 3 -5mm 3Prepare a 1.0% agarose solidifying agent by heating and dissolving it in advance. After cooling slightly, add it to a 1.5ml EP tube. Before the agarose solidifies, use tweezers to pick up the trimmed eyeball tissue and suspend it in the 1.0% agarose.

[0071] The solid block coated with 1.0% agarose solidifying agent in the above steps was immersed in 1.0% osmium tetroxide fixative (prepared with 0.1M phosphate buffer (pH 7.4)) and fixed at room temperature in the dark for 2.0 hours.

[0072] The tissue, after being fixed with 1.0% osmium tetroxide for 2.0 hours, was transferred to a 5.0 ml EP tube containing 2.0 ml of epoxy resin embedding medium (acetone:epoxy resin = 1:1) and slowly infiltrated at 37°C for 2.0-4.0 hours; then it was infiltrated overnight at 37°C with epoxy resin embedding medium (acetone:epoxy resin = 1:2); then it was slowly infiltrated with pure epoxy resin at 37°C for 5.0-8.0 hours. Finally, the pure epoxy resin was poured into the embedding plate, the sample was inserted into the embedding plate, and then it was oven-dried at 37°C overnight.

[0073] The embedding plates obtained in the above steps are polymerized in an oven at 60℃ for 48.0 hours, and the resin blocks are taken out for later use. The resin blocks are used for semi-thin sections with a thickness of 1.5 μm and for ultrathin sections with a thickness of 60-80 nm. The sections are retrieved using a 150-mesh copper mesh.

[0074] The ultrathin sections obtained in the above steps were divided into four groups. The treatment group adopted the technical solution of the present invention. Control group 1 used an EDTA-hydrogen peroxide composite solution with a pH of 9.0 (hydrogen peroxide content of 0.75%), control group 2 used a trichloroisocyanuric acid solution with a pH of 6.5 (trichloroisocyanuric acid concentration of 0.25wt%), and control group 3 used a 0.25% potassium permanganate solution. The melanin was decomposed according to the melanin decomposition solution formula of Example 1, and then treated with pre-decolorizing solution, decolorizing solution and rinsing solution.

[0075] Observation by transmission electron microscopy revealed that the treatment group is the technical solution of this invention. Figure 5 The top left (top left) area had the least melanin deposition and the lightest color, and its melanin removal effect was better than that of the control group.

[0076] Example 6

[0077] Semi-thin sections are a type of tissue sectioning that is thinner than paraffin sections and frozen sections, allowing for relatively direct observation of retinal melanin deposition and changes in retinal cell layer structure. Based on semi-thin sections of mouse eyeball tissue, using the semi-thin sections obtained in Example 5, toluidine blue staining was used to compare the effects of different melanin treatment methods on the structural integrity of retinal tissue. Control group 1 used an EDTA-hydrogen peroxide composite solution with a pH of 9.0 (0.75% hydrogen peroxide), control group 2 used a trichloroisocyanuric acid solution with a pH of 6.5 (0.25 wt% trichloroisocyanuric acid), and control group 3 used a 0.25% potassium permanganate solution.

[0078] Beneficial effects: The semi-thin sections processed by the technical solution of this invention show relatively less melanin deposition, no separation between the RPE cell layer and the optic nerve cell layer of the retina, and an intact retinal structure. Figure 6 (Top left) The retinal structures of control groups 1 (treated with EDTA-hydrogen peroxide composite solution at pH 9.0 (hydrogen peroxide content 0.75%)), 2 (treated with trichloroisocyanuric acid solution at pH 6.5 (trichloroisocyanuric acid concentration 0.25wt%)), and 3 (treated with 0.25% potassium permanganate solution) were incomplete, with significant separation between the RPE cell layer and the optic nerve cell layer. Figure 6 Top right Figure 6 Bottom left Figure 6 (Bottom right).

[0079] This technical solution discloses a method for preventing retinal detachment and melanin removal, comprising the steps of pre-fixation with a fixative, encapsulation with a solidifying agent, fixation with a fixative solution, embedding with an embedding agent, decomposing melanin with a melanin-decomposing solution, pre-decolorization with a pre-decolorizing solution, decolorization with a decolorizing solution, and elution with a rinsing solution. It provides an effective method for removing melanin by avoiding retinal RPE layer cell detachment, protein denaturation, and nucleic acid degradation. The main steps include using a pre-fixative to maintain the original state of tissue cells, a solidifying agent to maintain the integrity of the tissue structure, a melanin-decomposing solution composed of various extracts to decompose melanin particles, pre-decolorization and decolorization solutions to separate melanin, and a rinsing solution to wash away the decomposed melanin on the tissue surface. This method effectively decomposes melanin particles in tissue sections while ensuring the integrity of the retinal tissue structure and without damaging the surface proteins, nucleic acids, and cell structures of the tissue sections, maintaining the biological activity of various organelles on the section surface and meeting the needs of subsequent histochemical staining.

[0080] The above embodiments are only for illustrating the present invention and should not be regarded as limiting the scope of protection of the present invention. Within the knowledge of those skilled in the art, various changes can be made without departing from the spirit of this patent, such as changing the composition and ratio of melanin decomposition solution and solidifying agent, or changing the way tissue sections are obtained, such as semi-thin sections, ultrathin sections, frozen sections, etc.

Claims

1. A method for preventing melanin loss and retinal degeneration, characterized in that, It consists of the following steps: S1. Fixation with pre-fixative solution; S2, solidifying agent encapsulation and shaping; S3, fixation with a fixing solution; S4. Encapsulation with embedding agent; S5. Preparation of tissue sections; S6. Pretreatment of tissue sections: Dewaxing paraffin tissue sections to water or fixing frozen sections to water. S7. Decomposition of melanin with decomposition solution: Fix the slices from step S6 in a decolorization box, place the box on a decolorization shaker, immerse the slices in the melanin decomposition solution, pH 6.0-8.0, temperature 25℃-40℃, decolorization shaker speed 15 rpm-150 rpm, constant temperature and humidity in the dark for 2-48 hours; the decomposition solution consists of 10-30 parts of *Bacillus buddingus* extract, 15-25 parts of vitamins and their derivatives, 5-20 parts of arbutin and its derivatives, 10-15 parts of resveratrol and its derivatives, 3-15 parts of safflower seed oil and its derivatives, 1-10 parts of rhodioloside and its derivatives, 5-15 parts of passion fruit crude extract, 5-15 parts of red ginseng crude extract, and 5-15 parts of kiwifruit crude extract; *Bacillus buddingus* was deposited at the China General Microbiological Culture Collection Center on April 19, 2022, with accession number CGMCC. No. 40158, classified as Aureobasidium pullullans, deposited at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; S8, Pre-decolorizing solution; S9, decolorizing solution; S10, rinse with rinsing solution.

2. The method for preventing melanin loss and fading of melanin according to claim 1, characterized in that: The prefixation solution is a mixture of one or more of the following components, with a pH of 7.0: 1.0%-37.0% formaldehyde, 0.1%-25.0% glutaraldehyde, 1.0%-10.0% paraformaldehyde, 0.1%-1.0% osmium tetroxide, 0.01%-0.1% potassium permanganate, 0.01%-0.1% potassium dichromate, 5.0%-10.0% methanol, 5.0%-10.0% ethanol, 5.0%-10.0% acetic acid, 0.01%-0.05% mercuric chloride, and 1.0%-2.5% picric acid.

3. The method for preventing melanin loss and fading melanin according to claim 1, characterized in that: The solidifying agent is composed of multiple components, including 10-80 parts paraffin wax, 0.5-5 parts agarose, 0.5-15 parts epoxy resin, 0.5-10 parts collodion, 0.5-10 parts gelatin, 0.5-1 parts carbon wax, and 25-90 parts Optimal Cutting Temperature Compound (OCT).

4. The method for preventing melanin loss and fading of melanin according to claim 1, characterized in that: The embedding agent is composed of multiple components, including 10-80 parts paraffin, 0.5-5 parts agarose, 0.5-15 parts epoxy resin, 0.5-10 parts collodion, 0.5-10 parts gelatin, 0.5-1.0 parts carbon wax, and 25-90 parts Optimal Cutting Temperature Compound (OCT).

5. The method for preventing melanin loss and fading of melanin according to claim 1, characterized in that: The pre-decolorizing solution comprises one or more of the following components: magnesium chloride, magnesium sulfate, sodium chloride, sodium carbonate, ferric chloride, ferric oxide, ferric sulfide, ferrous sulfate, alkali-resistant penetrant OEP-70, and sodium dodecyl sulfonate, with a pH value of 7.0-8.

5.

6. The method for preventing melanin loss and fading of melanin according to claim 1, characterized in that: The decolorizing solution comprises one or more components selected from potassium hypochlorite, hypochlorous acid, sodium hypochlorite, potassium permanganate, dichromic acid, potassium dichromate, ozone, melatonin, glutathione, coenzyme Q10, hydrogen peroxide, sodium silicate, sodium dodecylbenzene sulfonate, sodium dodecyl sulfonate, alkali-resistant penetrant OEP-70, and 84 disinfectant, with a pH value of 7.0-8.

5.

7. The method for preventing melanin loss and fading of melanin according to claim 1, characterized in that: The rinsing solution comprises one or more components selected from penetrant, fluorescent whitening agent, oxalic acid, sodium bisulfite, sodium hyposulfite, and sodium thiosulfate, with a pH value of 6.5-7.9.

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