A tissue sample decoloring reagent and a method of processing a tissue

Abstract

The application discloses a kind of tissue sample decoloring reagent and the method for processing tissue, and the component of decoloring reagent includes hydrogen peroxide, anhydrous ethanol, polyethylene glycol octylphenyl ether and ethylenediaminetetraacetic acid.The method for processing tissue described in the application includes the tissue sample to be processed is immersed in decoloring reagent processing.Compared with prior art, the application uses hydrogen peroxide as the main decoloring component, removes endogenous autofluorescence such as hemoglobin, lipofuscin etc. in the interior of animal tissue by using its strong oxidizing property, and simultaneously removes residual pigment by using ethylenediaminetetraacetic acid and anhydrous ethanol for auxiliary decoloring, polyethylene glycol octylphenyl ether improves tissue permeability and enhances molecular penetration capacity.The application is suitable for various animal tissue samples, can prepare animal tissue sample thick section in a short time, and under the condition that various staining techniques are compatible and the structure of preserved tissue is complete, endogenous autofluorescence in tissue is removed to the greatest extent.

Description

Technical Field

[0001] This invention relates to the field of biological histology, specifically to a tissue sample decolorization reagent and a method for processing tissues. Background Technology

[0002] Animal tissue sample decolorization methods utilize various chemical reagents to remove endogenous autofluorescence from thick sections of animal tissue samples. By using different concentration ratios, endogenous pigments such as heme and lipofuscin inside the animal tissue can be effectively removed, reducing light absorption in optical microscopy imaging. This results in animal tissue samples that are free of endogenous autofluorescence and can be labeled by staining methods such as immunofluorescence labeling.

[0003] According to the specific implementation plan for animal tissue sample processing, the current section processing still focuses on thin sections of animal tissue a few micrometers thick and has not completely removed autofluorescence. The CUBIC removal scheme based on aqueous solvents for animal tissue section processing (Susaki, EA, et al., Cell, 2014.157(3):p.726-39) can remove endogenous autofluorescence in mouse tissue, human lungs, lymph nodes, etc. to a certain extent by using CUBIC-1 reagent, but it is not completely decolorized in tissues with high endogenous pigment, such as heart, liver, and spleen. As for decolorization based on organic solvents, 3DISICO (Ertürk, A., et al., Nature protocols, 2012.7(11):p.1983), in organs with high blood residue, such as spleen, bone marrow, and liver, heme is usually removed by bleaching or dissociation of heme, such as using acetone, but there is still no good organic reagent to remove endogenous pigments such as lipofuscin.

[0004] In summary, the CUBIC technique failed to effectively remove autofluorescence, especially for organs and tissues that could not be perfused and had a lot of residual blood, making it difficult to remove heme. It also failed to remove pigments such as lipofuscin in older tissues. The 3DISICO method used toxic organic solvents, was cumbersome to operate, and could not completely remove residual pigments such as lipofuscin. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a decolorizing reagent and treatment method for thick sections (50-600 micrometers) of animal tissue samples that is efficient, easy to operate and effectively compatible with multiple staining techniques.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] A tissue sample decolorization reagent comprising hydrogen peroxide, anhydrous ethanol, polyethylene glycol octylphenyl ether, and ethylenediaminetetraacetic acid.

[0008] Preferably, the tissue sample decolorizing reagent further includes water as a solvent.

[0009] Preferably, the tissue sample decolorizing reagent comprises, by volume percentage: 3-15% hydrogen peroxide, 3-8% anhydrous ethanol, 0.1-0.5% polyethylene glycol octylphenyl ether, 5-15% ethylenediaminetetraacetic acid, and the balance being water.

[0010] Preferably, the tissue sample decolorizing reagent has a molar ratio of hydrogen peroxide, ethylenediaminetetraacetic acid, and anhydrous ethanol of 1:2:1.

[0011] Preferably, the tissue sample decolorizing reagent comprises, by volume percentage: 3-8% hydrogen peroxide, 5% anhydrous ethanol, 0.1-0.5% polyethylene glycol octylphenyl ether, 10% ethylenediaminetetraacetic acid, and the balance being water.

[0012] Preferably, the tissue sample decolorizing reagent comprises, by volume percentage: 5% hydrogen peroxide, 5% anhydrous ethanol, 0.3% polyethylene glycol octylphenyl ether, 10% ethylenediaminetetraacetic acid, and the balance being water.

[0013] Preferably, the hydrogen peroxide concentration is adjusted according to the condition of the animal tissue. High concentrations of hydrogen peroxide (6%–10%) are required for aged tissue samples and tissues with high endogenous pigment content such as the heart, while low concentrations of hydrogen peroxide (3%–5%) are required for juvenile tissue samples and tissues with low endogenous pigment content such as the eyeball.

[0014] Preferably, the preparation method of the tissue sample decolorizing reagent of the present invention includes the following steps: mixing hydrogen peroxide aqueous solution, anhydrous ethanol, polyethylene glycol octylphenyl ether and ethylenediaminetetraacetic acid with water to obtain the tissue sample decolorizing reagent.

[0015] The present invention also proposes a method for treating tissues using the aforementioned tissue sample decolorizing reagent, comprising the following steps: immersing the tissue sample to be treated in the tissue sample decolorizing reagent for decolorization treatment.

[0016] Preferably, the decolorization treatment time is ≥1 hour.

[0017] Preferably, the decolorization treatment takes 12 to 36 hours.

[0018] Preferably, the decolorization process is carried out in a constant temperature shaker at 37°C, and the decolorization reagent is replaced every 4 to 12 hours.

[0019] Preferably, the thickness of the tissue sample to be processed is 50-600 μm.

[0020] Preferably, the tissue sample is an animal tissue sample.

[0021] Preferably, the decolorizing reagent of the present invention is stored at a low temperature (recommended 2℃~8℃) for later use.

[0022] Preferably, in the method of processing tissue, the animal tissue sample is embedded, and then the embedded animal tissue sample is continuously sliced ​​using a vibratory microtome or a paraffin microtome to obtain the tissue sample to be processed.

[0023] Preferably, after the thick sections of the embedded and fixed animal tissue samples are decolorized, they are then cleared, stained, matched with RIMS, and imaged. The clearing process can employ various clearing methods. The staining can employ various staining techniques, including immunofluorescence staining, DAPI staining, etc. The imaging can be performed using various optical microscopes, including laser confocal microscopy, two-photon microscopy, and laser sheet microscopy, etc.

[0024] The core objective of this invention is to propose a highly efficient, easy-to-use, and compatible decolorizing reagent for animal tissues, suitable for various staining methods. To achieve this objective, a decolorizing reagent was constructed comprising hydrogen peroxide, anhydrous ethanol, polyethylene glycol octylphenyl ether, and ethylenediaminetetraacetic acid (EDTA). The ratio of chemical reagents was adjusted according to the animal tissue sample. The concentration of hydrogen peroxide was the primary factor in regulating decolorization performance, while the concentrations of anhydrous ethanol, EDTA, and EDTA were adjusted as auxiliary factors. Specifically, this invention uses hydrogen peroxide as the main decolorizing reagent to effectively remove endogenous pigments such as heme and lipofuscin from the tissue. EDTA and anhydrous ethanol are used for auxiliary decolorization to remove residual pigments. EDTA improves tissue permeability and enhances molecular penetration. By simply immersing thick sections of animal tissue samples in the decolorizing reagent and treating them on a constant-temperature shaker for a certain period, difficult-to-remove endogenous autofluorescence such as heme and lipofuscin can be removed.

[0025] The advantages of this invention are as follows: This invention proposes a decolorizing reagent suitable for thick sections of animal tissue samples, which can effectively remove endogenous autofluorescence from animal tissue samples and play a role in removing background signals during subsequent staining and imaging processes. It is also compatible with various staining techniques and preserves the integrity of the tissue structure. A one-step decolorization method for animal tissue is employed, the reagent is simple to prepare, easy to store, and highly efficient. All reagents used in this invention are non-toxic or low-toxic, ensuring a high safety index during operation. This invention uses an appropriate concentration of ethylenediaminetetraacetic acid (EDTA) to achieve a pH of 9–11, effectively protecting the expression of immunofluorescence signals. It has a wide range of applications, effectively applicable to various animal tissue samples and suitable for animal tissue samples of different thicknesses. It is compatible with various clearing methods; compatible with various staining techniques, including immunofluorescence staining and FISH; and compatible with various imaging methods, including light slide microscopy and two-photon microscopy. The thickness of the animal tissue samples is typically tens of micrometers or more, unlike the few micrometers of traditional animal tissue sample sections. Attached Figure Description

[0026] Figure 1 The images show the decolorization effect of brain tumor tissue samples under different concentrations of decolorizing reagents and decolorization times in Example 1 of this invention.

[0027] Figure 2 These are autofluorescence images of brain tumor tissue samples before and after destaining in Example 2 of this invention;

[0028] Figure 3 This is a quantitative statistical chart comparing the autofluorescence intensity of brain tumor tissue samples without decolorization and after using different decolorization methods in Example 3 of the present invention.

[0029] Figure 4 These are neuronal staining images of brain tumor tissue in Example 4 of the present invention, before and after destaining using different destaining methods;

[0030] Figure 5 This is a quantitative statistical graph showing the effect of undecolorized brain tumor tissue and different decolorization methods on the intensity of leukocyte immunofluorescence expression in Example 4 of the present invention.

[0031] Figure 6 These are images of thick sections of pig brain tissue samples of different thicknesses treated with the decolorizing reagent in Example 5 of this invention.

[0032] Figure 7 These are high-resolution three-dimensional microscopic images of brain tumor tissue before and after decolorization in Example 6 of the present invention;

[0033] Figure 8 This is a high-resolution three-dimensional microscopic image of T lymphocytes after decolorization of brain tumor tissue in Example 7 of the present invention;

[0034] Figure 9 This is a comparison of the effects of different concentration ratios of the decolorizing reagent of the present invention on brain tissue after decolorization in Example 8 of the present invention;

[0035] Figure 10 This is a comparison of the effects of different concentration ratios of decolorizing agents on brain tumor tissue in Example 9 of the present invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0038] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0039] In the embodiments of the present invention, brain tissue and brain tumor tissue are decolorized, but the present invention can also be applied to other animal tissues.

[0040] Unless otherwise specified, the concentrations mentioned in this invention refer to the volume ratio of solute to solution.

[0041] The hydrogen peroxide of each concentration described in this invention is prepared by diluting commercially available high-concentration hydrogen peroxide aqueous solution.

[0042] Example 1

[0043] Brain tumor tissue samples obtained from a brain bank were embedded and fixed. The tissue samples were then sliced ​​into continuous sections 300 μm thick using a vibratory microtome. These sections were divided into three groups and destained using different concentrations of hydrogen peroxide. The destained reagent was a homogeneous mixture of hydrogen peroxide aqueous solution, ethylenediaminetetraacetic acid (EDTA), anhydrous ethanol, polyethylene glycol octylphenyl ether (PEG), and ultrapure water. The volume concentrations of hydrogen peroxide in the three groups were 3%, 5%, and 8%, respectively; the volume concentration of EDTA was 10%; the volume concentration of anhydrous ethanol was 5%; and the volume concentration of PEG was 0.3%. The tissue sections were immersed in the destained reagent and then placed in a 37°C constant-temperature shaker for destained treatment. The destained reagent was replaced every 6 hours. Images after destained treatment at 0h, 1h, 8h, and 18h ​​are shown below. Figure 1 As shown, by Figure 1It can be seen that after 1 hour of decolorization treatment, the three groups of decolorization reagents with different concentration ratios have a certain decolorization effect, and the best decolorization effect is achieved after 18 hours.

[0044] Example 2

[0045] In this embodiment, a brain tumor tissue sample was obtained from a brain bank. After embedding and fixing the tissue sample, it was cut into continuous sections with a thickness of 300 μm using a vibratory slicer. The continuous sections were divided into two groups and treated with the unbleached tissue and the debleaching reagent of this invention, respectively. The debleaching reagent was a homogeneous mixture of hydrogen peroxide aqueous solution, ethylenediaminetetraacetic acid (EDTA), anhydrous ethanol, polyethylene glycol octylphenyl ether (PEG), and ultrapure water, with a hydrogen peroxide volume concentration of 5%, EDTA volume concentration of 10%, anhydrous ethanol volume concentration of 5%, and PEG octylphenyl ether volume concentration of 0.3%. The continuous sections were immersed in the debleaching reagent and then placed in a constant temperature shaker at 37°C for 24 hours. The debleaching reagent needed to be replaced every 6 hours. After that, the tissue underwent clearing and refractive index matching treatment. Finally, the tissue was photographed using a light film imaging microscopy device. Figure 2 ,Depend on Figure 2 It can be seen that the autofluorescence of the tissue was effectively removed after the decolorization treatment.

[0046] Example 3

[0047] In this embodiment, a brain tumor tissue sample was obtained from a brain bank. After embedding and fixing the tissue sample, it was sliced ​​into continuous sections with a thickness of 300 μm using a vibratory microtome. The continuous sections were divided into three groups. One group was decolorized using the decolorizing reagent in Example 2 of this invention, following the steps in Example 2. Another group was prepared using CUBIC-1 reagent (25% wt ethylenediaminetetraacetic acid, 25% wt urea, 15% wt Triton X-100, and ultrapure water) from the CUBIC method. For example, to prepare 500g of CUBIC-1 reagent, 125g of urea, 156g of 80wt% ethylenediaminetetraacetic acid, and 144g of ultrapure water were mixed, heated, and after complete dissolution, the mixture was further stirred at room temperature, and 75g of Triton X-100 was added. X-100) was destaining according to the steps in Example 2. A blank control group was not destaining. The tissue was cleared, polymerized, and refractive index matched. Then, it was photographed using a light-film microscopic imaging device. The autofluorescence intensity of the obtained images was normalized, and statistical quantitative analysis was performed to obtain... Figure 3 , Figure 3 This indicates that the autofluorescence intensity of brain tumor tissue samples treated with the decolorizing reagent of the present invention is significantly lower than that of untreated tissue samples and also lower than that of samples decolorized with CUBIC-1 reagent.

[0048] Example 4

[0049] This embodiment uses a brain tumor tissue sample obtained from a brain bank. After undergoing the same embedding, slicing, grouping, destaining, and clearing processes as in Example 3 above, Nissl staining with Invitrogen NeuroTrace Nissl 500 / 525 green fluorescent dye was used to label neurons. Following polymerization and refractive index matching, images were captured using a light-film microscopy device. Figure 4 , Figure 4 (ab) shows the contrast of unbleached neuron staining; (a) shows the autofluorescence channel; (b) shows the neuron staining channel. Figure 4 (cd) shows the staining comparison of neurons after destaining with the destaining reagent of the present invention; (c) shows the autofluorescence channel; and (d) shows the neuron staining channel. Figure 4 (ef) shows the contrast of neuronal staining after destaining with CUBIC-1 reagent in the CUBIC method; (e) shows the autofluorescence channel; and (f) shows the neuronal staining channel. The fluorescence intensity of the imaging results was normalized and then subjected to statistical quantitative analysis, such as... Figure 5 This indicates that after decolorization using the decolorizing reagent of the present invention, the tissue sample can still retain the staining mark to the greatest extent while removing autofluorescence as much as possible.

[0050] Example 5

[0051] This example uses a pig brain tissue sample. The pig brain tissue sample was cut into sections with thicknesses of 50 μm and 600 μm. The sample was destained using the destaining reagent described in Example 2, following the steps outlined in Example 2. Afterward, the tissue underwent clearing, DAPI staining, polymerization, and refractive index matching. Finally, it was imaged using a light-film microscopy device. Figure 6 , Figure 6 (b) and (d) show the autofluorescence channel and DAPI staining imaging channel of a 50μm thick tissue sample after destaining. (b) shows the autofluorescence channel, and (d) shows the DAPI staining imaging channel. Figure 6 (a) and (c) show the autofluorescence channel and DAPI staining imaging channel of a 600 μm thick tissue sample after destaining. (a) shows the autofluorescence channel, and (c) shows the DAPI staining imaging channel. Figure 6 The results show that the destaining effect is good, the autofluorescence signal is low, the staining effect is good, and the signal-to-noise ratio is high.

[0052] Example 6

[0053] This embodiment uses brain tumor tissue samples obtained from a brain bank. After embedding and fixing the tissue sample, it was cut into continuous sections with a thickness of 300 μm using a vibratory microtome. The sample sections were divided into two groups: one group was not destained, and the other group was destained using the destained reagent in Example 2 according to the method in Example 2. Both groups of tissue samples were then cleared, stained with DAPI, polymerized, and subjected to refractive index matching. The original images were then captured using a light-film microscopic imaging device. The sections were 1×1×3.5 μm... 3 The original image at a certain resolution was reconstructed and stitched to a size of 4×4×4μm. 3 Voxel resolution images, then obtained by Imaris imaging. Figure 7 ,in Figure 7 (a) is the autofluorescence intensity map of the unbleached tissue. Figure 7 (b) is the autofluorescence intensity map of the tissue after destaining, from... Figure 7 It can be seen that the autofluorescence after decolorization is significantly lower than that before decolorization.

[0054] Example 7

[0055] This embodiment uses a brain tumor tissue sample obtained from a brain bank. After embedding and sectioning as described in Example 2, the tissue was destained using the destaining reagent described in Example 2, following the steps outlined in Example 2. The tissue was then cleared, immunofluorescence stained with Abcam Anti-CD3 antibody, polymerized, and subjected to refractive index matching. Finally, an immunofluorescence image of T lymphocytes was obtained using a light-film imaging microscopy device. Figure 8 As shown, Figure 8 (a) is an image of the autofluorescence channel. Figure 8 (b) is a staining image of T lymphocytes, from... Figure 8 It can be seen that the autofluorescence expression is low, and the T lymphocyte staining results are clear.

[0056] Example 8

[0057] In this embodiment, brain tissue samples were obtained from a brain bank. After embedding and fixation, serial sections (300 μm thick) were prepared. Four of these serial sections underwent different destaining treatments, one of which was not destaining. The hydrogen peroxide volume concentrations in the destaining reagents used for the other three sections were 2%, 8%, and 20%, respectively. All other components, concentrations, and processing steps were the same as in Example 2, resulting in imaging results. Figure 9 Among them, 9(a) has a hydrogen peroxide concentration of 2%, 9(b) has not been decolorized, 9(c) has a hydrogen peroxide concentration of 20%, and 9(d) has a hydrogen peroxide concentration of 8%. It can be seen that too low a concentration of hydrogen peroxide will result in incomplete decolorization, while too high a concentration of hydrogen peroxide will damage the tissue structure. An 8% hydrogen peroxide concentration can achieve a good decolorization effect while preserving the integrity of the tissue structure.

[0058] Example 9

[0059] In this embodiment, brain tumor tissue samples were obtained from brain banks. The whole tissue samples were embedded and fixed, then serially sliced ​​into 300μm thick sections using a vibratory microtome. Five consecutive sections underwent different destaining treatments, with the first section not being destained. The other four sections were immersed in a destaining reagent and then placed in a 37°C constant-temperature shaker for 24 hours, with the destaining reagent being replaced every 6 hours. Afterward, the tissue underwent clearing and refractive index matching treatments, and finally, images were obtained using a light-film imaging microscopy device. Figure 10 The decolorizing reagent consists of four components: the second decolorizing reagent is prepared by uniformly mixing hydrogen peroxide aqueous solution and ultrapure water, with a hydrogen peroxide volume concentration of 3%; the third decolorizing reagent is prepared by uniformly mixing hydrogen peroxide aqueous solution, ethylenediaminetetraacetic acid (EDTA), anhydrous ethanol, polyethylene glycol octylphenyl ether (PEG), and ultrapure water, with hydrogen peroxide volume concentration of 3%, EDTA volume concentration of 6%, anhydrous ethanol volume concentration of 3%, and PEG volume concentration of 0.3%; and the fourth decolorizing reagent is prepared by uniformly mixing hydrogen peroxide aqueous solution, EDTA, anhydrous ethanol, and PEG volume concentration of 0.3%. The first decolorizing agent is prepared by uniformly mixing ethyl phenyl ether with ultrapure water, and the volume concentrations of hydrogen peroxide (5%), ethylenediaminetetraacetic acid (EDTA) (10%), anhydrous ethanol (5%), and polyethylene glycol octyl phenyl ether (0.3%) are as follows: The second decolorizing agent is prepared by uniformly mixing hydrogen peroxide aqueous solution, EDTA, anhydrous ethanol, polyethylene glycol octyl phenyl ether, and ultrapure water, and the volume concentrations of hydrogen peroxide (8%), EDTA (10%), anhydrous ethanol (5%), and polyethylene glycol octyl phenyl ether (0.3%) are as follows: Figure 10 It is known that if hydrogen peroxide is used at too low a concentration without the addition of ethylenediaminetetraacetic acid and anhydrous ethanol, the decolorization effect will be poor. Appropriately increasing the concentration of components according to the tissue condition can effectively remove autofluorescence.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tissue sample decolorizing reagent, characterized by: The components include, by volume percentage: hydrogen peroxide 3-15%, anhydrous ethanol 3-8%, polyethylene glycol octylphenyl ether 0.1-0.5%, ethylenediaminetetraacetic acid 5-15%, and the balance being water; the thickness of the tissue sample is 50-600 μm; in the components, the molar ratio of hydrogen peroxide, ethylenediaminetetraacetic acid, and anhydrous ethanol is 1:2:

1.

2. The tissue sample destaining reagent of claim 1, wherein: The components include, by volume percentage: hydrogen peroxide 3-8%, anhydrous ethanol 5%, polyethylene glycol octylphenyl ether 0.1-0.5%, ethylenediaminetetraacetic acid 10%, and the balance being water.

3. The tissue sample destaining reagent according to any one of claims 1-2, wherein: The components include, by volume percentage: hydrogen peroxide 5%, anhydrous ethanol 5%, polyethylene glycol octylphenyl ether 0.3%, ethylenediaminetetraacetic acid 10%, and the balance being water.

4. A method of processing a tissue with the tissue sample decoloring reagent according to any one of claims 1 to 3, characterized by: The method comprises the following steps: The tissue sample to be treated is immersed in the tissue sample decolorizing reagent for decolorizing treatment.

5. The method of treating tissue with the tissue sample decolorizing reagent according to claim 4, characterized in that: The decolorizing treatment time is ≥1 h.

6. The method of treating tissue with the tissue sample decolorizing reagent according to claim 4, wherein: The decolorizing treatment is carried out in a 37℃ constant-temperature shaking table, and the decolorizing reagent is replaced every 4-12 h.

7. The method of treating tissue with the tissue sample decolorizing reagent according to any one of claims 4-6, wherein: The thickness of the tissue sample to be treated is 50-600 μm.

Images