CN116399674A - Tissue sample decoloring reagent and tissue treatment method - Google Patents
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention discloses a tissue sample decoloring reagent and a tissue treatment method. The method of treating tissue according to the present invention comprises immersing a tissue sample to be treated in a decolorizing agent. Compared with the prior art, the invention uses hydrogen peroxide as a main decoloring component, removes endogenous autofluorescence such as heme, lipofuscin and the like in animal tissues by utilizing the strong oxidizing property of the hydrogen peroxide, and simultaneously uses ethylenediamine tetraacetic acid and absolute ethyl alcohol for auxiliary decoloring to remove residual pigment, and polyethylene glycol octyl phenyl ether improves the tissue permeability and enhances the molecular penetrating capacity. The method is suitable for various animal tissue samples, can prepare thick sections of the animal tissue samples in a short time, and can remove endogenous autofluorescence of tissues to the greatest extent under the conditions of being compatible with various dyeing technologies and preserving tissue structure integrity.
Description
Technical Field
The invention relates to the technical field of biological histology, in particular to a tissue sample decoloring reagent and a tissue processing method.
Background
The animal tissue sample decoloring method is to remove endogenous autofluorescence in a thick section of the animal tissue sample by utilizing various chemical reagents, effectively remove endogenous pigments such as heme, lipofuscin and the like in the animal tissue by proportioning of different concentrations, reduce light absorption phenomenon in optical microscopic imaging, and further obtain the animal tissue sample without the endogenous autofluorescence marked by a staining method such as immunofluorescence marking and the like.
According to the specific embodiment of animal tissue sample treatment, the current slice treatment is still focused on thin slices of animal tissue that are several microns thick and the autofluorescence is not completely removed, and the removal protocol CUBIC (Susaki, E.A., et al, cell,2014.157 (3): p.726-39) suitable for animal tissue slice treatment based on aqueous solvents can remove endogenous autofluorescence of mouse tissue, lung, lymph node, etc. of human tissue to some extent by using CUBIC-1 reagent, but on high endogenous pigment tissue such as heart, liver, spleen, etc. is not completely decolorized. In contrast, 3DISICO (Ert ulk, A., et al, nature protocols,2012.7 (11): p.1983) decolorization, which is based on organic solvents, is used to remove heme in organs with high blood residues, such as spleen, bone marrow and liver, by bleaching or dissociating heme, such as acetone, but endogenous pigments such as lipofuscin are not removed with good organic reagents.
By combining the above technologies, the decolorization effect of the CUBIC technology cannot effectively remove autofluorescence, especially for organ tissues which cannot be perfused and have more blood residues, heme in the organ tissues cannot be removed easily, and pigments such as lipofuscin in senile tissues cannot be removed, while the 3DISICO method uses toxic organic solvents, is complex in operation, and cannot remove residual pigments such as full-fat limonin the organ tissues.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a decoloring reagent and a processing method which are efficient, easy to operate and effectively compatible with various dyeing technologies and are suitable for thick sections (50-600 microns) of animal tissue samples.
The invention solves the technical problems by the following technical means:
a tissue sample decoloring reagent comprises hydrogen peroxide, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ethylenediamine tetraacetic acid.
Preferably, the tissue sample decolorizing agent further comprises solvent water.
Preferably, the tissue sample decolorizing agent comprises the following components in percentage by volume: 3-15% of hydrogen peroxide, 3-8% of absolute ethyl alcohol, 0.1-0.5% of polyethylene glycol octyl phenyl ether, 5-15% of ethylenediamine tetraacetic acid and the balance of water.
Preferably, the tissue sample decolorizing reagent comprises the following components in mole ratio of 1:2:1.
preferably, the tissue sample decolorizing agent comprises the following components in percentage by volume: 3-8% of hydrogen peroxide, 5% of absolute ethyl alcohol, 0.1-0.5% of polyethylene glycol octyl phenyl ether, 10% of ethylenediamine tetraacetic acid and the balance of water.
Preferably, the tissue sample decolorizing agent comprises the following components in percentage by volume: 5% of hydrogen peroxide, 5% of absolute ethyl alcohol, 0.3% of polyethylene glycol octyl phenyl ether, 10% of ethylenediamine tetraacetic acid and the balance of water.
Preferably, the concentration of hydrogen peroxide is adjusted according to the condition of animal tissues, high-concentration hydrogen peroxide is required for high-content endogenous pigment tissues such as senile tissue samples, hearts and the like (6% -10%), and low-concentration hydrogen peroxide is required for low-content endogenous pigment tissues such as juvenile tissue samples, eyeball tissues and the like (3% -5%).
Preferably, the preparation method of the tissue sample decolorizing reagent of the present invention comprises the following steps: and uniformly mixing the aqueous hydrogen peroxide solution, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ethylenediamine tetraacetic acid with water to obtain the tissue sample decoloring reagent.
The invention also provides a method for treating tissue by using the tissue sample decolorizing reagent, which comprises the following steps: immersing the tissue sample to be treated in the tissue sample decolorizing reagent for decolorizing treatment.
Preferably, the time of the decoloring treatment is more than or equal to 1h.
Preferably, the time of the decoloring treatment is 12 to 36 hours.
Preferably, the material is placed in a shaking table with constant temperature of 37 ℃ for decolorization, and the decolorization reagent is replaced every 4 to 12 hours.
Preferably, the thickness of the tissue sample to be treated is 50-600 μm.
Preferably, the tissue sample is an animal tissue sample.
Preferably, the decolorizing agent of the present invention is stored at low temperature (recommended 2-8 ℃) for use.
Preferably, in the method of treating a tissue, an animal tissue sample is subjected to embedding treatment, and then the embedded animal tissue sample is subjected to continuous slicing by using a vibration microtome or a paraffin microtome to obtain a tissue sample to be treated.
Preferably, the thick section of the embedded and fixed animal tissue sample is decolorized, and then is subjected to tissue transparentization, dyeing, RIMS matching and imaging; the transparentizing treatment can adopt various transparentizing methods; the staining may employ a variety of staining techniques including immunofluorescent staining, DAPI staining, and the like; the imaging can be performed using a variety of optical microscopes, including laser confocal microscopes, two-photon microscopes, laser slab microscopes, and the like.
The invention aims at providing a decoloring reagent which is efficient, easy to operate and effectively compatible with various dyeings and is suitable for animal tissues, and around the purpose, a decoloring reagent with hydrogen peroxide, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ethylenediamine tetraacetic acid as components is constructed; according to the condition of animal tissue samples, adjusting the proportion of chemical reagents; the main property of the decolorization is that the concentration of hydrogen peroxide is regulated, and the concentration of absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ethylene diamine tetraacetic acid is regulated as an auxiliary; specifically, hydrogen peroxide is used as a main decolorizing reagent, endogenous pigments such as heme, lipofuscin and the like in tissues are effectively removed, and simultaneously ethylenediamine tetraacetic acid and absolute ethyl alcohol are used for auxiliary decolorizing to remove residual pigments, polyethylene glycol octyl phenyl ether improves tissue permeability and enhances molecular penetration capacity, and endogenous autofluorescence which is difficult to remove such as heme, lipofuscin and the like can be removed by simply soaking thick sections of animal tissue samples into the decolorizing reagent and processing the thick sections under a constant-temperature shaking table for a plurality of times.
The invention has the advantages that: the invention provides a decoloring reagent suitable for thick sections of animal tissue samples, which can effectively remove endogenous autofluorescence of the animal tissue samples, can play a role in removing background signals in the subsequent dyeing marking and imaging processes, and can be compatible with various dyeing technologies and preserve tissue structure integrity; the animal tissue decoloring method with one-step treatment is adopted, the reagent is simple to manufacture, the storage is easy, and the treatment is efficient. The reagents used in the invention are nontoxic or low-toxicity reagents, and the safety index of the operation process is high; the invention adopts ethylenediamine tetraacetic acid with proper concentration, so that the pH value is 9-11, and the expression of immunofluorescence signals is effectively protected; the application range is wide, and the method is effectively applicable to various animal tissue samples and can be used for animal tissue samples with different thicknesses; is compatible with various transparentization methods; the fluorescent dye is compatible with various dyeing technologies, and immunofluorescence dyeing, FISH and the like can be effectively compatible; the imaging method is compatible with various imaging means, and the light sheet microscopic imaging and the two-photon microscopic imaging can be effectively compatible; the animal tissue samples are each tens of microns thick or more, differing from the several microns of conventional animal tissue sample slices.
Drawings
FIG. 1 is a graph showing the decoloring effect of brain tumor tissue samples of example 1 under different concentrations of decoloring agent and decoloring times;
FIG. 2 is an autofluorescence image of brain tumor tissue samples without and after decolorization in example 2 of the present invention;
FIG. 3 is a graph showing comparative quantification of the autofluorescence intensity of brain tumor tissue samples without decolorizing and after using different decolorizing methods in example 3 of the present invention;
FIG. 4 is a graph showing the staining of neurons after not decoloring brain tumor tissue and using different decoloring methods in example 4 of the present invention;
FIG. 5 is a graph showing the quantitative statistics of the influence of the non-decolorized brain tumor tissue and different decolorization methods on the immunofluorescence expression intensity of leucocytes in example 4 of the present invention;
FIG. 6 is a thick slice image of a sample of porcine brain tissue treated with decolorizing agents of example 5 of the present invention;
FIG. 7 is a high resolution three-dimensional microscopic image of brain tumor tissue before and after decoloring in example 6 of the present invention;
FIG. 8 is a high resolution three-dimensional microscopic image of T lymphocytes after decolorization of brain tumor tissue in example 7 of the present invention;
FIG. 9 is a graph showing the comparison of the effects of the decolorizing brain tissue with the decolorizing agents of the present invention at different concentration ratios in example 8 of the present invention;
FIG. 10 is a graph showing the comparison of the effects of decolorizing brain tumor tissues with decolorizing agents of different concentration ratios in example 9 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
In the embodiment of the invention, the brain tissue and the brain tumor tissue are decolorized, but the invention can be applied to other animal tissues as well.
The concentration of the invention is the volume ratio of solute to solution under the condition of no special description.
The hydrogen peroxide of each concentration is prepared by diluting and proportioning commercial high-concentration hydrogen peroxide aqueous solution.
Example 1
The method comprises the steps of embedding and fixing a brain tumor tissue sample obtained from a brain library application, cutting the tissue sample into 300 mu m-thick serial slices by using a vibration slicer, dividing the tissue sample slices into three groups, respectively using decolorizing agents with different hydrogen peroxide concentrations to perform decolorizing treatment, wherein the decolorizing agents are formed by uniformly mixing hydrogen peroxide aqueous solution, ethylenediamine tetraacetic acid, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ultrapure water, the three groups of decolorizing agents have hydrogen peroxide volume concentrations of 3%, 5% and 8%, the ethylenediamine tetraacetic acid volume concentration is 10%, the absolute ethyl alcohol volume concentration is 5%, the polyethylene glycol octyl phenyl ether volume concentration is 0.3%, immersing the tissue sample slices in the decolorizing agents at a constant temperature of 37 ℃ for decolorizing treatment, and replacing the decolorizing agents once every 6 hours, wherein the decolorizing agents with different concentrations have certain decolorizing effects after the decolorizing treatment of 0h, 1h, 8h and 18h, and the best decolorizing effect is achieved after the decolorizing treatment of a shaking table after the decolorizing treatment of 1h as shown in fig. 1.
Example 2
In this example, after embedding and fixing a tissue sample obtained from a brain library application, cutting the tissue sample into 300 μm thick serial sections by using a vibration microtome, dividing the serial sections into two groups, respectively performing tissue non-decoloring treatment and decoloring reagent treatment according to the invention, wherein the decoloring reagent is prepared by uniformly mixing hydrogen peroxide aqueous solution, ethylenediamine tetraacetic acid, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ultrapure water, the volume concentration of hydrogen peroxide is 5%, the volume concentration of ethylenediamine tetraacetic acid is 10%, the volume concentration of absolute ethyl alcohol is 5%, the volume concentration of polyethylene glycol octyl phenyl ether is 0.3%, immersing the serial sections of the sample in the decoloring reagent, then placing the serial sections in a constant temperature shaking table at 37 ℃ for 24 hours, replacing the decoloring reagent every 6 hours, performing transparentization and refractive index matching treatment, and finally shooting by using a light sheet illumination microscopic imaging device to obtain fig. 2, and as can be seen from fig. 2, the autofluorescence of the tissue is effectively removed after the decoloring treatment.
Example 3
In this example, after embedding and fixing the tissue sample, the tissue sample is cut into 300 μm thick serial sections by using a vibrating microtome, the serial sections are divided into three groups, one group is decolorized by using the decolorizing reagent in example 2 according to the procedure in example 2, the other group is prepared by using the CUBIC-1 reagent (25% wt. ethylenediamine tetraacetic acid, 25% wt. urea, 15% wt. Triton X-100 and ultrapure water) in the CUBIC method, for example, 500g of CUBIC-1 reagent is prepared, 125g of urea and 156g of 80% wt. ethylenediamine tetraacetic acid are mixed with 144g of ultrapure water, heated and mixed, after complete dissolution, the mixture is further stirred at room temperature, 75 g of Triton X-100) is added for decolorizing treatment according to the procedure in example 2, one group of blank controls is not decolorized, the tissue is subjected to transparentization, polymerization and refractive index matching treatment, then the obtained image is photographed by using a light piece illumination microscopy imaging device, the fluorescence intensity of the obtained image is normalized, and the fluorescence intensity of the obtained image is subjected to statistics of FIG. 3, and the fluorescence intensity of the sample is significantly lower than that of the tissue sample is obtained after the fluorescence sample is subjected to the spontaneous tumor sample analysis, and the fluorescence sample is not subjected to the fluorescence sample is subjected to the fluorescence sample analysis 3.
Example 4
This example is a brain tumor tissue sample obtained from a brain library application, after the same embedding, slicing, grouping, decoloring and transparentizing treatments as in example 3 above, neurons were labeled with Invitrogen NeuroTrace Nissl/525 green fluorescent dye, and then polymerized and index-matched, and a photograph was taken using a light-sheet illumination microscopy imaging device to obtain fig. 4, fig. 4 (a-b) being a non-decoloring neuron staining comparative, (a) being an autofluorescent channel, (b) being a neuron staining channel, fig. 4 (c-d) being a neuron staining comparative after decoloring with the decoloring agent of the present invention, (c) being an autofluorescent channel, (d) being a neuron staining channel, fig. 4 (e-f) being a post-coloring neuron comparative with the CUBIC-1 agent in the CUBIC method, (e) being an autofluorescent channel, and (f) being a neuron staining channel. The fluorescence intensity of the imaging result is normalized, and through statistical quantitative analysis, as shown in fig. 5, it is illustrated that after the decolorization by the decolorizing agent of the present invention, the tissue sample can still retain the staining mark to the greatest extent under the condition that autofluorescence is removed as much as possible.
Example 5
The present example is a pig brain tissue sample cut into sections with thickness of 50 μm and 600 μm, decolorizing treatment was performed by using the decolorizing reagent in example 2 according to the procedure in example 2, then the tissue was subjected to transparentization, DAPI staining, polymerization and refractive index matching treatment, and then the light-sheet illumination microscopic imaging device was used to photograph to obtain fig. 6, fig. 6 (b, d) shows an autofluorescence channel and a DAPI staining imaging channel after decolorizing a 50 μm thick tissue sample, (b) shows an autofluorescence channel, (d) shows a DAPI staining imaging channel, fig. 6 (a, c) shows an autofluorescence channel and a DAPI staining imaging channel after decolorizing a 600 μm thick tissue sample, (a) shows an autofluorescence channel and a DAPI staining imaging channel, and the results in fig. 6 show that the decolorizing effect is good, the autofluorescence signal is low, and the staining effect is good, and the signal to noise ratio is high.
Example 6
In this example, a brain tumor tissue sample obtained from a brain library application was fixed by embedding, cutting the tissue sample into 300 μm thick serial sections using a vibrating microtome, dividing the sections into two groups, one group was not decolorized, one group was decolorized by the decolorizing reagent of example 2 according to the method of example 2, and the two groups were subjected to transparentization, DAPI staining, polymerization and refractive index matching, and then photographed by a light-sheet illumination microscopy imaging device to obtain an original image, and 1×1×3.5 μm 3 Original image of resolution reconstructing and splicing into 4X 4 μm 3 The voxel resolution size image is imaged by using Imaris to obtain fig. 7, wherein fig. 7 (a) is an image of the tissue autofluorescence intensity after decolorization, and fig. 7 (b) is an image of the tissue autofluorescence intensity after decolorization, and as can be seen from fig. 7, the autofluorescence after decolorization is significantly lower than that of the tissue after decolorization.
Example 7
In this example, a brain tumor tissue sample obtained from a brain library application was subjected to the same embedding and slicing as in example 2, and then subjected to the decoloring treatment according to the procedure of example 2 using the decoloring reagent of example 2, and then the tissue was subjected to the steps of transparentizing, immunofluorescent staining with an Abcam Anti-CD3 antibody, polymerization and refractive index matching treatment, and then an immunofluorescent imaging image of T lymphocytes was obtained by photographing with a light-sheet illumination microscopic imaging device, as shown in fig. 8, fig. 8 (a) was an autofluorescence channel imaging image, and fig. 8 (b) was a T lymphocyte staining imaging image, and as can be seen from fig. 8, the autofluorescence expression was low and the T lymphocyte staining result was clear.
Example 8
In this example, all brain tissue samples obtained from the brain library application were embedded and fixed, serial sections (thickness 300 μm) were obtained, four serial sections were subjected to different decoloring treatments, one of the sections was not subjected to the decoloring treatment, the volume concentration of hydrogen peroxide in the other three sections was 2%, 8%, 20%, and the other components, concentrations and treatment steps were the same as those in example 2, and an imaging result was shown in fig. 9, in which 9 (a) was 2% hydrogen peroxide concentration, 9 (b) was not subjected to the decoloring treatment, 9 (c) was 20% hydrogen peroxide, 9 (d) was 8% hydrogen peroxide, hydrogen peroxide at an excessively low concentration could cause incomplete decoloring effect, hydrogen peroxide at an excessively high concentration could damage the tissue structure, and 8% hydrogen peroxide concentration was compatible with good decoloring effect under the condition of preserving the tissue integrity structure.
Example 9
In this embodiment, the whole tissue sample is embedded and fixed, the tissue sample is cut into 300 μm thick slices by a vibration microtome, wherein five slices are continuously processed without decoloring, the other four slices are immersed in decoloring reagent and then are processed in a constant temperature shaking table at 37 ℃ for 24 hours, decoloring reagent is replaced every 6 hours, then tissue is subjected to transparentization and refractive index matching treatment, finally, the tissue is photographed by using an optical-sheet illumination microscopic imaging device to obtain fig. 10, wherein the second decoloring reagent is formed by uniformly mixing hydrogen peroxide aqueous solution with ultrapure water, the hydrogen peroxide volume concentration is 3%, the third decoloring reagent is formed by uniformly mixing hydrogen peroxide aqueous solution, ethylenediamine tetraacetic acid, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether with ultrapure water, the fourth decoloring reagent is prepared by uniformly mixing a hydrogen peroxide aqueous solution, ethylenediamine tetraacetic acid, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ultrapure water, wherein the hydrogen peroxide volume concentration is 3%, the ethylenediamine tetraacetic acid volume concentration is 6%, the absolute ethyl alcohol volume concentration is 3%, the polyethylene glycol octyl phenyl ether volume concentration is 0.3%, the fourth decoloring reagent is prepared by uniformly mixing a hydrogen peroxide aqueous solution, ethylenediamine tetraacetic acid, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ultrapure water, the hydrogen peroxide volume concentration is 5%, the ethylenediamine tetraacetic acid volume concentration is 10%, the absolute ethyl alcohol volume concentration is 5%, the polyethylene glycol octyl phenyl ether volume concentration is 0.3%, the fifth decoloring reagent is prepared by uniformly mixing a hydrogen peroxide aqueous solution, ethylenediamine tetraacetic acid, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ultrapure water, and the hydrogen peroxide volume concentration is 8%, the ethylenediamine tetraacetic acid volume concentration is 10%, the absolute ethyl alcohol volume concentration is 5%, the volume concentration of the polyethylene glycol octyl phenyl ether is 0.3%; as is clear from fig. 10, too low a concentration of hydrogen peroxide, without adding ethylenediamine tetraacetic acid or absolute ethanol, tends to cause poor decoloring effect, and autofluorescence can be effectively removed by appropriately increasing the component concentration according to the tissue condition.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A tissue sample decolorizing reagent, characterized in that: the components of the composition comprise hydrogen peroxide, absolute ethyl alcohol, polyethylene glycol octyl phenyl ether and ethylenediamine tetraacetic acid.
2. The tissue sample decolorizing reagent of claim 1, wherein: the components also comprise solvent water.
3. The tissue sample decolorizing reagent of claim 1, wherein: the components of the composition comprise the following components in percentage by volume: 3-15% of hydrogen peroxide, 3-8% of absolute ethyl alcohol, 0.1-0.5% of polyethylene glycol octyl phenyl ether, 5-15% of ethylenediamine tetraacetic acid and the balance of water.
4. The tissue sample decolorizing reagent of claim 1, wherein: the molar ratio of hydrogen peroxide to ethylenediamine tetraacetic acid to absolute ethyl alcohol is 1:2:1.
5. a tissue sample decolorizing reagent according to any one of claims 1-3, wherein: the components of the composition comprise the following components in percentage by volume: 3-8% of hydrogen peroxide, 5% of absolute ethyl alcohol, 0.1-0.5% of polyethylene glycol octyl phenyl ether, 10% of ethylenediamine tetraacetic acid and the balance of water.
6. A tissue sample decolorizing reagent according to any one of claims 1-3, wherein: the components of the composition comprise the following components in percentage by volume: 5% of hydrogen peroxide, 5% of absolute ethyl alcohol, 0.3% of polyethylene glycol octyl phenyl ether, 10% of ethylenediamine tetraacetic acid and the balance of water.
7. A method of treating tissue with a tissue sample decolorizing reagent of any one of claims 1-6, wherein the method comprises: the method comprises the following steps: immersing the tissue sample to be treated in the tissue sample decolorizing reagent for decolorizing treatment.
8. The method of treating tissue with the tissue sample decolorizing agent of claim 7, wherein: the time of the decoloring treatment is more than or equal to 1h.
9. The method of treating tissue with the tissue sample decolorizing agent of claim 7, wherein: placing the mixture in a shaking table with constant temperature of 37 ℃ for decoloring, and replacing decoloring reagent every 4 to 12 hours.
10. The method of treating tissue with the tissue sample decolorizing agent of any one of claims 7-9, wherein: the thickness of the tissue sample to be treated is 50-600 μm.
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