Method for preventing melatonin from falling off

Abstract

The method comprises the following steps: fixing with a pre-fixing solution, wrapping and forming with a solid agent, fixing with a fixing solution, embedding with an embedding agent, decomposing melanin with a melanin decomposing solution, pre-decoloring with a pre-decoloring solution, decoloring with a decoloring solution, and eluting with a rinsing solution. The invention provides a melanin removing method capable of effectively avoiding retina RPE layer cell exfoliation, protein denaturation and nucleic acid degradation. The method mainly comprises the following steps: keeping the original state of tissue cells by adopting a pre-fixing solution, keeping the complete tissue structure by adopting a solid agent, decomposing melanin particles by adopting a melanin decomposing solution composed of various extracts, separating melanin by adopting a pre-decoloring solution and a decoloring solution, eluting the melanin decomposed on the surface of the tissue by adopting a rinsing solution, and the like. On the premise that the retina tissue structure is intact and protein, nucleic acid and cell structures on the surface of the tissue section are not damaged, melanin particles in the tissue section are effectively decomposed, the biological activity of various organelles on the surface of the section is kept, and the requirements of subsequent chemical staining of various tissues are met.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a method for preventing melatonin from being desorbed, and further relates to a method for effectively avoiding retinal RPE layer cell desorption, cell damage and nucleic acid degradation during melanin removal. Background Art

[0002] Melanin is a unique type of biomacromolecule pigment formed by the oxidative polymerization of polyphenols or indole monomers. It is widely distributed in many fungi, plants, animals and microorganisms. It is classified into artificial melanin and natural melanin according to its source. Natural melanin is divided into eumelanin, allomelanin and pheomelanin according to the different chemical precursors used in its biosynthetic pathway. The color of eumelanin is mainly black or brown. From the perspective of molecular structure, it mainly contains nitrogen and does not contain sulfur. It is formed through a series of oxidative polymerization reactions. Isomelanin is black in color, and its main feature is that there is no nitrogen in elemental analysis. It is widely present in plants and some microorganisms. Pheomelanin contains nitrogen and sulfur elements and is slightly reddish brown in color.

[0003] Most melanins are hydrophobic and negatively charged macromolecules. They are poorly soluble in aqueous solutions and common organic solvents. They are very stable deposited pigments. They are usually not black under a microscope, but yellow-brown or tan. They do not contain iron but contain sulfur. They are insoluble in water and organic solvents. They can be dissolved after a long period of exposure to strong alkali and decolorized by strong oxidants. Melanin is usually found in the basal cells of the squamous epithelium of the skin, the hair bulbs, the iris, choroid and ciliary body of the eyeball, and the substantia nigra of the midbrain. Some tumor cells also contain melanin-like substances.

[0004] Melanocytes produce melanin, which is mainly stored in the melanin granules in the cytoplasm, which can easily cause the cell structure of the section of the tissue section to be blurred, and interfere with and affect the results of subsequent chemical staining such as hematoxylin staining and immunohistochemical staining. In particular, the retinal structure of the eyeball tissue is relatively fragile, and its RPE layer cells are easily separated from the retina in the liquid, causing denetwork, affecting subsequent experimental observations. The present invention mainly utilizes pre-fixing solution fixation, solid agent packaging and forming, fixing solution fixation, embedding agent embedding, melanin decomposition solution decomposition of melanin, pre-decolorization with pre-decolorization solution, decolorization with decolorization solution and elution with rinsing solution. Under the premise of not damaging other proteins and cell structures in the tissue section, different melanin decomposition methods are provided according to different purposes, so that the melanin in the tissue section changes from brown to colorless, and the cell structure is clearly displayed, meeting the needs of various subsequent chemical staining. Summary of the invention

[0005] The purpose of the present invention is to provide a method for removing melanin particles that prevents retinal detachment, has little cell damage, has high pigment removal efficiency, and is not easy to degrade nucleic acids (including various nucleic acids such as DNA\RNA\cDNA), and specifically provides a melanin removal method that can meet various types of sections (such as paraffin sections, frozen sections, semi-thin sections and ultra-thin sections). The method mainly adopts the steps of pre-fixing solution fixation, solid agent wrapping and forming, fixing solution fixation, embedding agent embedding, melanin decomposition solution decomposition, pre-decolorization with pre-decolorization solution, decolorization with decolorization solution and elution with rinsing solution. Under the premise of ensuring the integrity of the retinal structure and not damaging the exposed cells, proteins, nucleic acids, etc. on the surface of the tissue, the melanin particles are decomposed, and the cell structure is kept intact, the protein activity is not lost, and the nucleic acid is not degraded, so as to meet the needs of various subsequent chemical staining and molecular operations.

[0006] In order to achieve the above objectives, the following technical solutions are mainly adopted.

[0007] Fixation with pre-fixative solution: fresh tissues were immediately placed in tissue pre-fixative solution for fixation for more than 24 hours after dissection; the pre-fixative solution consisted 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 ingredients were configured in proportion to form a mixed solution with a pH of 7.0.

[0008] The solid agent is wrapped and formed, and the tissue is taken out from the pre-fixing solution. The target part of the tissue is trimmed and cut flat with a scalpel. The size of the trimmed tissue is determined according to the type of slice. Semi-thin slices and ultra-thin slices require epoxy resin. The tissue size is generally 2mm 3 -5mm 3 , tissue size of OCT embedded sections and paraffin embedded sections 3mm 3 -10mm 3 ); prepare the solid by heating and dissolving in advance, add the solid into a 1.5 ml EP tube after it is slightly cooled, pick up the trimmed tissue with forceps and suspend and wrap it in the solid before the solid solidifies, cool it in a -20°C freezer, take it out and trim the embedded block after the solid embedding solidifies; the solid consists of 10-80 parts of paraffin, 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), and one or more of the above ingredients are configured into the solid according to proportion.

[0009] Fixation with fixative: the tissue block wrapped with solid agent is immediately put into tissue fixative and fixed again for more than 24 hours; 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. One or more of the above ingredients are configured in proportion to form a mixed solution with a pH of 7.0.

[0010] Tissue embedding: Tissue blocks that are secondary fixed with fixative are further embedded in different embedding agents according to different experimental purposes. The embedding agent consists of 10-80 parts of paraffin, 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 ingredients are configured in proportion to form a pre-embedding agent.

[0011] Preparation of tissue sections, paraffin tissue sections: place the trimmed embedded block on the paraffin slicer and slice it, 5μm thick. The slices float on the 40℃ warm water of the spreader to flatten the tissue, pick up the slide, and bake the slices in a 60℃ oven. After the water-dried wax is baked, take it out and store it at room temperature for later use; frozen tissue sections: fix the embedding table on the slicer, first roughly cut the tissue surface and trim it flat before starting to slice, the slice thickness is 8-10μm, place a clean slide flat on the cut tissue slice, and then stick the tissue on the slide. After the slice is labeled, store it at -20℃ for later use; semi-thin and ultra-thin sections: place the embedded tissue block in a 60℃ oven for polymerization for 48h, take out the embedded block and slice it on the ultra-thin slicer, the thickness of the semi-thin section is generally about 1.5μm, the thickness of the ultra-thin section is 60-80nm, and the 150-mesh Fanghua membrane copper mesh is used to pick up the slice.

[0012] Pre-treatment of tissue sections: dewax paraffin tissue sections to water: put the sections into environmentally friendly dewaxing solution I for 10 minutes - environmentally friendly dewaxing solution II for 10 minutes - environmentally friendly dewaxing solution III for 10 minutes - anhydrous ethanol I for 5 minutes - anhydrous ethanol II for 5 minutes - anhydrous ethanol III for 5 minutes - distilled water for washing; fix frozen sections to water: dry the frozen sections at room temperature, bake in a 37°C oven for 10-20 minutes, place them in methanol for 20 minutes, and wash them in PBS (pH 7.4) on a decolorizing shaker for 3 times, each time for 5 minutes; semi-thin sections and ultra-thin sections: proceed directly to the subsequent steps.

[0013] The melanin decomposition liquid decomposes melanin, and the melanin decomposition liquid mainly consists of 10-30 parts of Aspergillus or Saccharomyces cerevisiae extract or budding short pedunculate enzyme 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 salidroside 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 the above components are mixed in proportion, the pH is adjusted to 6.0-8.0, the tissue sections are fixed in a decolorization box, the decolorization box is placed on a decolorization shaker, the above melanin decomposition solution is added, the tissue sections are immersed in the melanin decomposition solution, the decomposition temperature is 25-40°C, the decolorization shaker speed is 15-150rpm, and the decomposition is carried out in a constant temperature and humidity dark environment for 2-48 hours.

[0015] Preferably, the budding pullulanase extract is derived from the fermentation broth of the budding pullulanase strain EP1001, which was deposited in the China General Microbiological Culture Collection Center (CGMCC for short) on April 19, 2022; the deposit number is CGMCC No. 40158, and the classification name is Aureobasidium pullulans, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] Preferably, purine or pyrimidine and its derivatives include one or more of adenosine phosphate, adenosine diphosphate, adenosine triphosphate, uridine phosphate, uridine diphosphate, uridine triphosphate, guanosine phosphate, guanosine diphosphate, guanosine triphosphate, cytidine phosphate, cytidine diphosphate and cytidine triphosphate, and one or more of cyclic adenosine phosphate, cyclic adenosine diphosphate, cyclic adenosine triphosphate, cyclic uridine phosphate, cyclic uridine diphosphate, cyclic uridine triphosphate, cyclic guanosine monophosphate, cyclic guanosine diphosphate, cyclic guanosine triphosphate, cyclic cytidine phosphate, cyclic cytidine diphosphate and cyclic cytidine triphosphate.

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

[0018] Preferably, the 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 decolorized, and the pre-decolorizing solution is composed of various cations and anions, especially divalent and trivalent metal ions and non-metal examples, including but not limited to a mixture of one or more of magnesium chloride, magnesium sulfate, sodium chloride, sodium carbonate, ferric chloride, ferric oxide, ferric sulfide, ferrous sulfate, alkali-resistant penetrant OEP-70, sodium dodecyl sulfate, etc. The pH value of the mixed pre-decolorizing solution is 7.0-8.5.

[0021] Preferably, the contents of the main components in the pre-decolorization solution are: 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, iron oxide 0.001g / L-0.1g / L, iron 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, sodium dodecyl sulfate 0.1g / L-0.5g / L.

[0022] The decolorizing solution includes a mixture of one or more of 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, 84 disinfectant, etc. The pH value of the mixed decolorizing solution is 7.0-8.5.

[0023] Preferably, the contents of the main components in the decolorizing solution are: 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, 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] Rinsing with a rinsing solution, the rinsing solution mainly includes a mixture of one or more of a penetrant, a fluorescent whitening agent, oxalic acid, sodium bisulfite, sodium hyposulfite or sodium thiosulfate, and the pH value of the mixed rinsing solution is 6.5-7.9.

[0025] Preferably, the contents of the main components in the rinsing liquid are: alkali-resistant penetrant OEP-70 1.5g / L-2.8g / L, fluorescent brightener 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of hematoxylin-eosin chemical staining of mouse eye paraffin sections. The left side is the staining result before demelaninization, and the right side is the staining result after demelaninization. It can be seen with the naked eye that the melanin particles in the right picture have almost disappeared.

[0028] Figure 2 The results of immunohistochemical staining of VEGF protein in paraffin sections of mouse eyeballs. The left side is the immunohistochemical result before demelaninization, and the right side is the immunohistochemical result after demelaninization. It can be seen with the naked eye that the brown-yellow color on the right side has basically disappeared, indicating that the brown-yellow color on the left side is a false positive result produced by the melanin reaction.

[0029] Figure 3 Comet gel electrophoresis was used to detect intracellular DNA damage. The upper left figure shows the technical solution of the present invention, the upper right figure shows an EDTA-hydrogen peroxide composite solution with a pH value of 9.0 (hydrogen peroxide content of 0.75%), the lower left figure shows a trichloroisocyanuric acid solution with a pH value of 6.5 (the concentration of trichloroisocyanuric acid is 0.25wt%), and the lower right figure shows a 0.25% potassium permanganate solution.

[0030] Figure 4 Flow cytometry was used to detect cell apoptosis. The upper left image shows the technical solution of the present invention, the upper right image shows an EDTA-hydrogen peroxide composite solution with a pH value of 9.0 (hydrogen peroxide content of 0.75%), the lower left image shows a trichloroisocyanuric acid solution with a pH value of 6.5 (the concentration of trichloroisocyanuric acid is 0.25wt%), and the lower right image shows a 0.25% potassium permanganate solution.

[0031] Figure 5 Transmission electron microscopy was used to observe the removal of melanin in ultrathin sections of mice. The upper left is the technical solution of the present invention, the upper right is an EDTA-hydrogen peroxide composite solution with a pH value of 9.0 (hydrogen peroxide content of 0.75%), the lower left is a trichloroisocyanuric acid solution with a pH value of 6.5 (the concentration of trichloroisocyanuric acid is 0.25wt%), and the lower right is a 0.25% potassium permanganate solution.

[0032] Figure 6 Semi-thin sections were used to observe the structural integrity of the mouse retina and the removal of melanin. The upper left is the technical solution of the present invention, the upper right is an EDTA-hydrogen peroxide complex solution with a pH value of 9.0 (hydrogen peroxide content of 0.75%), the lower left is a trichloroisocyanuric acid solution with a pH value of 6.5 (the concentration of trichloroisocyanuric acid is 0.25wt%), and the lower right is a 0.25% potassium permanganate solution. DETAILED DESCRIPTION Example 1

[0033] Ocular melanocytes include the ocular pigment epithelium, scleral (corneal) epithelium, melanocytes in the brown layer of the sclera and the uveal stroma, as well as melanocytes in the ocular appendages (eyelids and conjunctiva). Therefore, it is most appropriate to use the hematoxylin-eosin chemical staining effect of mouse eye pathological tissue sections to confirm the decomposition effect of the melanin particles of the technical solution.

[0034] After dissection, fresh intact eyeball tissue was immediately placed in 4.0% paraformaldehyde fixative with a pH of 7.5 for pre-fixation for more than 24 hours. The volume of the pre-fixative was 10 times the volume of the tissue.

[0035] Remove the tissue from the pre-fixing solution and use a scalpel to trim the target area of ​​tissue flat. The size of the trimmed tissue is 5mm. 3 -9mm 3 ; Prepare 1.0% agarose solid by heating and dissolving in advance. After the 1.0% agarose is slightly cooled, add it into a 1.5ml EP tube. Before the 1.0% agarose solidifies, pick up the trimmed eyeball tissue with tweezers and suspend it in the 1.0% agarose and cool it in a -20℃ freezer. After solidification, take it out and trim the embedding block.

[0036] Place the melted wax into the embedding frame, and before the wax solidifies, place the tissue wrapped in the solid agent into the embedding frame and label it accordingly. Cool it in a -20° freezer, and after the wax solidifies, remove the wax block from the embedding frame and trim it.

[0037] Place the wax block trimmed in the above steps on a paraffin slicer to slice 5 μm thick. Float the slices on 40°C warm water on the slicer to flatten the tissue, pick up the slides, bake the slices in a 60°C oven, dry the wax in water, and then take them out and store them at room temperature for later use.

[0038] The sections obtained in the above steps were sequentially placed in environmentally friendly dewaxing liquid I for 10 min-environmentally friendly dewaxing liquid II for 10 min-environmentally friendly dewaxing liquid III for 10 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-anhydrous ethanol III for 5 min-distilled water for washing.

[0039] The tissue sections obtained in the above steps are fixed in a decolorization box, which is placed on a decolorization shaker and immersed in a melanin decomposition solution containing 25 parts of budding short pedunculate extract, 25 parts of vitamin C, 15 parts of arbutin, 3 parts of resveratrol, 7 parts of safflower seed oil, 7 parts of salidroside, 5 parts of passion fruit crude extract, 8 parts of red ginseng crude extract, and 5 parts of kiwi crude extract. The decomposition temperature is 28° C., the decolorization shaker speed is 100 rpm / hr, and the decomposition is carried out in a constant temperature and humidity dark environment for 48 hours.

[0040] In the above step, the tissue sections were decomposed in the dark for 48 hours, the decomposition solution was discarded, and the sections were immersed in a pre-decolorization 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 1-2 times, 5 minutes / time, and the decolorization box containing the tissue sections enzymatically hydrolyzed by the enzymatic solution was immersed in the pre-decolorization 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 a constant temperature and humidity for 18 hours.

[0041] The tissue sections pre-decolorized for 18 hours in the above step were discarded from the pre-decolorization solution, and were soaked 1-2 times with a decolorization solution containing 30 ml / L 27.5% hydrogen peroxide, 2.0 g / L sodium silicate, 6.0 g / L sodium dodecyl sulfate, and 1.5 g / L alkali-resistant penetrant OEP-70 at a pH of 8.0 for 5 minutes each time, and then the tissue sections were immersed in the decolorization solution for decolorization treatment, the decolorization temperature was 40°C, the decolorization shaker speed was 100 rpm / hr, and the decolorization was carried out in a constant temperature and humidity dark environment for 30 minutes.

[0042] The tissue sections decolorized for 30 minutes in the above step, discard the decolorizing solution, soak in a rinsing solution containing 5.0 g / L alkali-resistant penetrant OEP-70, 0.3 g / L fluorescent brightener, 3.0 g / L oxalic acid, 4.0 g / L sodium bisulfite with a pH value of 7.0 for 2-3 times, 10 minutes each time, and then rinse with the rinsing solution, the rinsing temperature is 35°C, the rinsing shaker speed is 80 rpm / hr, and the constant temperature and humidity rinsing is performed 2-3 times, 15 minutes each time.

[0043] After decolorization, the tissue sections were placed in hematoxylin staining solution for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue re-staining solution, and rinsed with running water. The sections were placed in 95% alcohol for dehydration for 1 minute, and stained in eosin staining solution for 15 seconds. The sections were placed in 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 - xylene II for 2 minutes, transparent, sealed with neutral gum, and observed with a microscope. The cell nucleus was blue and the cytoplasm was red ( Figure 1 ).

[0044] Beneficial effect: after being treated with the scheme of the present invention, the melanin particles almost disappear ( Figure 1 On the right side), the cell structure of the eyeball tissue is clear, and the complete cell structure of the retina, optic nerve, and iris can be clearly seen. Example 2

[0045] Immunohistochemical staining is based on the reaction between antigens and antibodies. The activity and content of antigens in tissues are the key factors affecting the immunohistochemical effect. In the process of removing melanin particles, strong acids, strong alkalis and strong oxidants can destroy the exposed protein structure in tissue sections and change the physical and chemical properties of proteins, and even cause protein denaturation and inactivation, affecting the subsequent binding effect of exogenous immunohistochemical antibodies and target proteins.

[0046] Based on the paraffin sections of mouse eyeball tissue, the melanin particles in the tissue are decomposed by gentle melanin decolorization conditions, reducing the interference of melanin on immunohistochemical positive signals and reducing the occurrence of false positives. The experimental steps are the same as those in Example 1. After rinsing, the slices are subjected to antigen repair, blocking of endogenous peroxidase, 3% BSA serum blocking, incubation with VEGF protein primary antibody, incubation with HRP-labeled goat anti-mouse secondary antibody, DAB color development, hematoxylin counterstaining of cell nuclei, dehydration and sealing, and then placed under a white light microscope for result interpretation. Hematoxylin stains cell nuclei in blue, and DAB shows positive expression in brown ( Figure 2 ).

[0047] Beneficial effect: after the melanin is treated by the immunohistochemical step, it generally appears brownish yellow, and the positive result after the immunohistochemical antibody and antigen are combined is also shown as brownish yellow, so melanin can interfere with the positive result of immunohistochemistry of tissue sections; in this embodiment, after the tissue sections are treated by melanin decolorization, there is almost no brownish yellow positive result after immunohistochemical staining with VEGF antibody ( Figure 2 On the right side), while the tissue sections that have not been depigmented by melanin have a darker brown-yellow result after immunohistochemical staining with VEGF antibody ( Figure 2 Left side), indicating that the brown-yellow color on the left is a false positive result produced by the melanin reaction. Therefore, demelaninizing the tissue section can effectively reduce the interference of false positive signals.

[0048] Example 3

[0049] The comet assay, also known as the single-cell gel electrophoresis assay, can effectively detect and quantitatively analyze the degree of single-strand and double-strand gap damage in DNA in cells. When various endogenous and exogenous DNA damage factors induce cell DNA chain breaks, their supercoiled structures are destroyed. Under the action of the cell lysate, membrane structures such as the cell membrane and nuclear membrane are destroyed, and the proteins, RNA and other components in the cell are diffused into the electrolyte, while the nuclear DNA cannot enter the gel due to its large molecular weight and remains in place. In order to better demonstrate the technical advantages of the present application, based on mouse eyeball tissue, the effects of different melanin treatment methods on the damage of nucleic acids (DNA\RNA\cDNA, etc.) in tissue sections are compared. The preliminary experimental steps are the same as in Example 1. After the tissues rinsed with the rinse solution are digested with 2.5% trypsin for 2 hours, the digested cells are subjected to the comet gel electrophoresis experiment. The specific experimental steps are operated according to the instructions of the comet electrophoresis kit (Article No.: C2041M, Brand: Beyotime), and the specific steps are as follows.

[0050] Preparation of the first layer of glue: drop 180 μl of 1% normal melting point agarose NMA (dissolved in phosphate buffer PBS without Ca2+ and Mg2+) preheated to 45°C onto the frosted surface of the slide that has been preheated in the same way, quickly cover it with a clean coverslip, and place it at 4°C for 10 min to solidify.

[0051] Preparation of the second layer of glue: Take 100 μl of cell suspension and 150 μl of 0.8% low melting point agarose LMA (dissolved in phosphate buffer PBS without Ca2+ and Mg2+) and mix them at 37°C, then gently peel off the coverslip, drop the cell-containing LMA onto the first layer of glue, immediately cover it with a clean coverslip, and solidify it at 4°C for 20 min.

[0052] Preparation of the third layer of gel: Remove the slide, quickly drop 100 μL of 0.5% low melting point agarose at 37°C on the second layer of agarose, cover with a slide, and solidify at 4°C for 20 min. 3. Lysis: Remove the slide and immerse the slide in the newly prepared 4°C lysis solution for 2 h.

[0053] Unwinding: Remove the slide from the lysis solution, wash off excess salt in distilled water, and air dry. Pour the newly prepared electrophoresis buffer into the electrophoresis tank, covering the slide by 0.25 cm, cover it with a lid, and place it in the electrophoresis buffer for 30 min.

[0054] Electrophoresis: At room temperature, adjust the buffer level and run electrophoresis at 300 mA and 25 V for 15 min.

[0055] Washing and staining: After electrophoresis, place the slides in a dish and rinse with neutralization buffer for 30 min. After neutralization, stain the slides with 50 μL-100 μL acridine orange solution (EB) for 10 min and rinse with distilled water for more than 2 times.

[0056] Result observation: After staining, use a fluorescence microscope to examine as soon as possible. Leaving too long will cause fluorescence fading. The above process should be performed in the dark to avoid additional DNA damage.

[0057] The treatment group adopted the technical solution of the present invention ( Figure 3 Upper left), control group 1 used EDTA-hydrogen peroxide complex solution with a pH value of 9.0 (hydrogen peroxide content 0.75%) ( Figure 3 Upper right), control group 2 used a trichloroisocyanuric acid solution with a pH value of 6.5 (the concentration of trichloroisocyanuric acid was 0.25wt%) (( Figure 3 Lower left)), Control group 3 was treated with 0.25% potassium permanganate solution ( Figure 3 Lower right), after decolorization of melanin, single cell suspensions were prepared for comet gel electrophoresis experiments in the four groups.

[0058] Comet Assay Software Pect (CASP 1.2.3 beta 1)) image analysis software was used to analyze the comet images. According to the DNA content (TDNA%) in the comet tail in the tailing cells, the degree of cell DNA damage was divided into 5 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: Table 1 Statistics of DNA damage degree detected by comet gel electrophoresis under different melanin removal conditions

[0059] TailDNA% is the percentage of tailing DNA in the total DNA. Three independent cells were selected for statistical results in each group. AV is the average value of TailDNA% of the three cells in each group, and SD stands for standard deviation.

[0060] Finally, it was found that the degree of DNA damage in the treatment group was grade 1 (Table 1, Figure 3 The DNA damage levels of the other three groups were all grade 3 (Table 1, Figure 3 Top right, Figure 3 Lower left, Figure 3Lower right), it can be concluded that the technical solution of the present invention is a method that causes minimal damage to nucleic acids (DNA or RNA, etc.).

[0061] Example 4 In order to further verify the effect of the technical effect of the present invention on cell structure, the effects of different melanin treatment methods on cell damage were compared based on mouse eyeball tissue. The preliminary experimental steps were the same as in Example 1. The treatment group adopted the technical solution of the present invention ( Figure 4 Upper left), control group 1 used EDTA-hydrogen peroxide complex solution with a pH value of 9.0 (hydrogen peroxide content 0.75%) ( Figure 4 Upper right), control group 2 used a trichloroisocyanuric acid solution with a pH value of 6.5 (the concentration of trichloroisocyanuric acid was 0.25wt%) (( Figure 4 Lower left)), Control group 3 was treated with 0.25% potassium permanganate solution ( Figure 4 Lower right), after melanin decolorization and rinsing with rinsing solution, the tissue was digested with 2.5% trypsin for 2 hours, and the digested cells were detected for apoptosis using flow cytometry.

[0062] Table 2 Statistics of cell apoptosis detected by flow cytometry

[0063] Grouping Proportion of late apoptosis (%) Proportion of premature apoptosis (%) Total percentage of apoptosis (%) Treatment 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

[0064] The results showed that the treatment group (Table 2, Figure 4 The cell apoptosis rate of the upper left sample is the lowest, the cell damage is minimal, and the integrity of the cell structure in the tissue section can be better protected.

[0065] Example 5

[0066] Ultrathin sections are the main technical method for observing subcellular structures in tissues, and can more intuitively observe the deposition of melanin in cells. In order to further verify the technical effect of the present invention, ultrathin sections of mouse tissues were used as the basis to compare the effects of different melanin treatment methods on the removal of melanin in ultrathin sections.

[0067] After dissection, fresh intact eyeball tissue was immediately placed in 2.5% glutaraldehyde fixative with a pH of 7.5 for pre-fixation for more than 24 hours. The volume of the pre-fixative was 10 times the volume of the tissue.

[0068] Remove the tissue from the pre-fixing solution and use a scalpel to trim the target area of ​​tissue flat. The size of the trimmed tissue is 3mm. 3 -5mm 3 ; Prepare 1.0% agarose solid by heating and dissolving in advance, add it to a 1.5ml EP tube after it cools slightly, and before the agarose solidifies, pick up the trimmed eyeball tissue with tweezers and suspend it in the 1.0% agarose.

[0069] The solid block coated with 1.0% agarose solid in the above step was immersed in 1.0% osmium tetroxide fixative (prepared with 0.1 M phosphate buffer (pH 7.4)) and fixed at room temperature in the dark for 2.0 hr.

[0070] The above tissues fixed with 1.0% osmium tetroxide for 2.0 hours were transferred to 5.0ml EP tubes containing 2.0ml epoxy resin embedding agent (acetone: epoxy resin = 1:1), and slowly infiltrated at 37℃ for 2.0-4.0 hours; then infiltrated with epoxy resin embedding agent (acetone: epoxy resin = 1:2) at 37℃ overnight; then infiltrated slowly with pure epoxy resin at 37℃ for 5.0-8.0 hours, and finally pure epoxy resin was poured into the embedding plate, and the sample was inserted into the embedding plate and placed in an oven at 37℃ overnight.

[0071] The embedded plate obtained in the above steps was polymerized in an oven at 60°C for 48.0 hours, and the resin block was taken out for use; the resin block was used for semi-thin sections with a thickness of 1.5um, and the resin block was used for ultra-thin sections with a thickness of 60-80nm, and the sections were scooped out using a 150-mesh square copper mesh.

[0072] The ultrathin section tissue obtained in the above steps was divided into four groups, wherein the treatment group adopted the technical solution of the present invention, the control group 1 adopted an EDTA-hydrogen peroxide composite solution with a pH value of 9.0 (hydrogen peroxide content of 0.75%), the control group 2 adopted a trichloroisocyanuric acid solution with a pH value of 6.5 (the concentration of trichloroisocyanuric acid was 0.25wt%), and the control group 3 adopted a 0.25% potassium permanganate solution. The melanin was decomposed according to the melanin decomposition solution formula of Example 1, and the pre-decolorization solution decolorization, decolorization solution decolorization and rinsing solution elution steps were treated.

[0073] After transmission electron microscopy observation, the treated group, i.e., the technical solution of the present invention ( Figure 5 The upper left) has the least melanin deposition and the lightest color, and the melanin removal effect is better than that of the control group.

[0074] Example 6 Semi-thin sections are a type of tissue sectioning method that is thinner than paraffin sections and frozen sections, and can relatively intuitively observe the retinal melanin deposition and changes in the retinal cell layer structure. Based on the semi-thin sections of mouse eyeball tissue, the semi-thin sections obtained in the experimental steps of Example 5 were used to compare the effects of different melanin treatment methods on the integrity of retinal tissue structure by toluidine blue staining, wherein the control group 1 used an EDTA-hydrogen peroxide composite solution with a pH value of 9.0 (hydrogen peroxide content of 0.75%), the control group 2 used a trichloroisocyanuric acid solution with a pH value of 6.5 (the concentration of trichloroisocyanuric acid was 0.25wt%), and the control group 3 used a 0.25% potassium permanganate solution.

[0075] Beneficial effect: after the semi-thin sections are treated with the technical solution of the present invention, melanin deposition is relatively small, the retinal RPE cell layer and the optic nerve cell layer are not separated, and the retinal structure is intact ( Figure 6 Left upper), the retinal structures of the control group 1 (using EDTA-hydrogen peroxide complex solution with a pH value of 9.0 (hydrogen peroxide content of 0.75%)), the control group 2 (trichloroisocyanuric acid solution with a pH value of 6.5 (trichloroisocyanuric acid concentration of 0.25wt%)) and the control group 3 (using 0.25% potassium permanganate solution) were incomplete, and the RPE cell layer and the optic nerve cell layer were obviously separated ( Figure 6 Top right, Figure 6 Lower left, Figure 6 lower right).

[0076] The present technical scheme discloses a method for preventing melatonin from being de-reticulated, comprising the steps of fixing with a pre-fixing solution, wrapping and forming with a solid agent, fixing with a fixing solution, embedding with an embedding agent, decomposing melanin with a melanin decomposing solution, pre-decolorizing with a pre-decolorizing solution, decolorizing with a decolorizing solution, and eluting with a rinsing solution. A method for removing melanin that effectively avoids the de-reticulation of retinal RPE layer cells, protein denaturation, and nucleic acid degradation is provided. The pre-fixing solution is mainly used to maintain the original state of tissue cells, the solid agent maintains the integrity of the tissue structure, and various extracts are used to form a melanin decomposing solution to decompose melanin particles, the pre-decolorizing solution and the decolorizing solution are used to separate melanin, and the rinsing solution is used to elute the melanin decomposed on the tissue surface. Under the premise of ensuring the integrity of the retinal tissue structure and not damaging the proteins, nucleic acids, and cell structures on the surface of the tissue sections, the melanin particles in the tissue sections are effectively decomposed, the biological activity of various organelles on the surface of the sections is maintained, and the needs of various subsequent tissue chemical staining are met.

[0077] The above embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of protection of the present invention. Various changes can be made within the knowledge of ordinary technicians in this field without departing from the purpose of this patent, such as changing the components and proportions of the melanin decomposition solution and the solid agent, or changing the method of obtaining tissue sections, such as semi-thin sections, ultra-thin sections, frozen sections, etc.

Claims

1. A method for preventing melatonin from being removed from the network, comprising the steps of fixing with a pre-fixing solution, wrapping with a solid agent to form, fixing with a fixing solution, embedding with an embedding agent, decomposing melanin with a melanin decomposing solution, pre-decolorizing with a pre-decolorizing solution, decolorizing with a decolorizing solution, and eluting with a rinsing solution. It is characterized by: The solid agent is composed of one or more of paraffin wax 10-80 parts, agarose 0.5-5 parts, epoxy resin 0.5-15 parts, collodion 0.5-10 parts, gelatin 0.5-10 parts, carbon wax 0.5-1 parts and Optimal Cutting Temperature Compound (OCT) 25-90 parts; the embedding medium is composed of paraffin wax 10-25 parts, agarose 1-10 parts, epoxy resin 0.2-15 parts, collodion 0.5-20 parts, gelatin 3.5-10 parts, carbon wax 0.3-1.0 parts and Optimal Cutting Temperature Compound (OCT) 25-90 parts. The melanin decomposition liquid is composed of one or more ingredients in 35-65 parts of OCT; the melanin decomposition liquid is composed of one or more ingredients in 10-30 parts of budding short-stalk enzyme extract, 3-15 parts of purine or pyrimidine and its derivatives, 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 crude extract, 1-10 parts of salidroside crude extract, 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.

2. The method for preventing melatonin from being lost according to claim 1, characterized in that: The pre-decolorizing solution is composed of magnesium chloride, magnesium sulfate, sodium chloride, sodium carbonate, ferric chloride, ferric oxide, ferric sulfide, ferrous sulfate, alkali-resistant penetrant OEP-70 and sodium dodecyl sulfate. The pre-decolorizing solution is a mixture of one or more of the above ingredients, and the pH value of the mixed pre-decolorizing solution is 7.0-8.

5.

3. The method for preventing melatonin from being lost according to claim 1, characterized in that: The contents of the components in the pre-decolorization solution are respectively 0.1g / L-1.0g / L of magnesium chloride, 0.05g / L-0.25g / L of magnesium sulfate, 1.5g / L-3.0g / L of sodium chloride, 0.06g / L-1.36g / L of sodium carbonate, 0.006g / L-0.02g / L of ferric chloride, 0.001g / L-0.1g / L of iron oxide, 0.25g / L-1.0g / L of iron sulfide, 5.0g / L-8.0g / L of ferrous sulfate, 1.5g / L-2.8g / L of alkali-resistant penetrant OEP-70, and 0.1g / L-0.5g / L of sodium dodecyl sulfate.

4. The method for preventing melatonin from being lost according to claim 1, characterized in that: The decolorizing solution includes potassium hypochlorite, hypochlorous acid, sodium hypochlorite, potassium permanganate, dichromic acid, potassium dichromate, ozone, melatonin, glutathione, coenzyme Q10, hydrogen peroxide, sodium dodecylbenzene sulfonate, sodium dodecylsulfonate, alkali-resistant penetrant OEP-70, and 84 disinfectant. The pH value of the pre-decolorizing solution after mixing one or more of the above ingredients is 7.0-8.

5.

5. A method for preventing melatonin from being lost according to claim 1 or 4, characterized in that: The contents of the components in the decolorizing solution are respectively 0.05g / L-0.2g / L of potassium hypochlorite, 0.03g / L-0.18g / L of hypochlorous acid, 0.05g / L-0.2g / L of sodium hypochlorite, 0.001g / L-0.01g / L of potassium permanganate, 0.002g / L-0.015g / L of dichromic acid, 0.009g / L-0.01g / L of potassium dichromate, 0.0005g / L-0.001g / L of ozone, 0.21g / L-0.5g / L of melatonin, 2.5g / L-5.0g / L of glutathione, 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.

6. The method for preventing melatonin from being lost according to claim 1, characterized in that: The rinsing liquid mainly includes alkali-resistant penetrant OEP-70, fluorescent whitening agent, oxalic acid, sodium bisulfite, sodium hyposulfite, sodium thiosulfate, etc. The pH value of the mixed rinsing liquid is 6.5-7.

9.

7. A method for preventing melatonin from being lost according to claim 1 or 6, characterized in that: The contents of the components in the rinsing liquid are respectively 1.5g / L-2.8g / L of alkali-resistant penetrant OEP-70, 3.5g / L-4.8g / L of fluorescent whitening agent, 2.5g / L-2.9g / L of oxalic acid, 0.95g / L-5.0g / L of sodium bisulfite, 0.5g / L-3.0g / L of sodium hyposulfite or 2.5g / L-5.0g / L of sodium thiosulfate.

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