An ex vivo biological tissue staining buffer based on electric field-chemical closed-loop pH dynamic regulation and application

Abstract

The application discloses an ex vivo biological tissue staining buffer based on electric field-chemical closed-loop pH dynamic regulation and application thereof. The staining buffer is mainly formed by dissolving the following components in deionized water: buffer: one or a combination of two of tris(hydroxymethyl)aminomethane and 3-(cyclohexylamino)-1-propanesulfonic acid; surfactant: Triton X-100, Tween 20; pH regulator: glucose; and the ex vivo biological tissue staining buffer is arranged in a direct current electric field device. The reagent components of the application are simple, and are suitable for various staining molecules such as antibodies and fluorescent dyes, and provide an electric field staining technical scheme with high efficiency, mildness and controllability for high-throughput three-dimensional pathology, whole organ mapping and accurate diagnosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological tissue marking, and particularly relates to an ex vivo biological tissue staining buffer based on electric field-chemical closed-loop pH dynamic regulation and application. BACKGROUND

[0002] The existing biological tissue marking is still mainly based on passive diffusion of staining molecules. This method is limited by random Brownian motion and concentration gradient, and the marking time is often counted by days. More seriously, the dense extracellular matrix, high-fat myelin sheath and natural pigment of the tissue itself jointly form a "triple barrier", so that the dye molecules are exponentially distributed within a millimeter depth--excessive consumption on the surface and serious shortage in the deep. As a result, the signal is uneven, the signal-to-noise ratio is unbalanced, and false images are formed which are difficult to remove.

[0003] In order to break through the diffusion limit, the electric field driven staining technology emerges as the times require. However, the existing electric field buffer generally adopts the formula of borate / Tris / TritonX-100, and the pH range is fixed at 9.0-10.0. In this strong alkaline environment, the histidine, tyrosine and lysine residues of the antibody / antigen binding site are quickly deprotonated, the electrostatic attraction drops cliff-like (Ka drops by 1-2 orders of magnitude), resulting in a quadratic decay of the binding efficiency with depth; at the same time, the continuous high pH also causes the deformation of the tissue and the loss of the antigen epitope, forming the paradox of "the more you use the electric field, the worse it gets". In short, the alkaline barrier has become a fundamental technical bottleneck restricting the uniform staining of centimeter level. SUMMARY

[0004] In order to solve the problem of deep staining failure caused by "alkaline barrier", the application provides an electric field-chemical closed-loop self-regulating pH staining buffer. The buffer uses glucose as a "programmable acid generator", which quantitatively releases formic acid / acetic acid in a 20-60 V cm-1 direct current electric field through cascade oxidation, so that the pH of the system falls from 9.0±0.1 to 7.2-7.8 within 6 hours, thereby restoring the antibody-antigen electrostatic attraction (Ka increases by 1-2 orders of magnitude) in real time, and realizing the uniform, non-destructive and rapid marking of centimeter level tissue.

[0005] In order to achieve the above purpose, the technical scheme adopted by the application is:

[0006] I. Composition of the ex vivo biological tissue staining buffer based on electric field-chemical closed-loop pH dynamic regulation

[0007] The ex vivo biological tissue staining buffer is mainly formed by dissolving buffer, surfactant and pH regulator in deionized water, the pH regulator is glucose, and the ex vivo biological tissue staining buffer is arranged in a direct current electric field device.

[0008] The molar concentration of the buffer in the ex vivo biological tissue staining buffer is 10-20 mM, and the buffer is one or a combination of the other two of tris(hydroxymethyl)aminomethane, 3-(cyclohexylamine)-1-propanesulfonic acid. The function of the buffer is to maintain the pH environment required by the staining molecules (antibodies, fluorescent dyes, etc.), prevent the denaturation of target molecules, provide uniform conductivity, and ensure uniform electric field distribution.

[0009] The volume concentration of the surfactant in the ex vivo biological tissue staining buffer is 0.05-0.5% v / v, and the surfactant is one or a combination of the other two of TritonX-100, Tween20. The function of the surfactant is to reduce the non-specific binding of the staining molecules, reduce the background noise, and improve the signal-to-noise ratio.

[0010] The molar concentration of the pH adjuster in the ex vivo biological tissue staining buffer is 30-50 mM.

[0011] II. Application of the ex vivo biological tissue staining buffer based on the dynamic regulation of pH in the electric field-chemical closed loop

[0012] Immunostaining detection and three-dimensional imaging of ex vivo biological tissues. The ex vivo biological tissue includes an ex vivo human brain, kidney, liver, or complete animal organ, and the ex vivo biological tissue in the ex vivo biological tissue staining buffer is subjected to three-dimensional imaging to obtain results including one or a combination of staining depth, tissue swelling rate, and fine structure of the ex vivo biological tissue.

[0013] III. Electric field-chemical closed loop dynamic regulation method applied to the ex vivo biological tissue staining buffer

[0014] The ex vivo biological tissue staining buffer is placed in a direct current electric field device, the direct current electric field device is turned on, and the glucose is quantitatively converted into C1-C3 short-chain carboxylic acids through cascade oxidation under the driving of the direct current electric field, so that the pH of the ex vivo biological tissue staining buffer itself decreases within 5-7 hours.

[0015] The initial pH value of the buffer in the ex vivo biological tissue staining buffer is set to 9.0±0.1, and the pH decrease rate is linearly related to the glucose concentration. The glucose in the ex vivo biological tissue staining buffer is quantitatively converted into C1-C3 short-chain carboxylic acids through cascade oxidation under the driving of the direct current electric field, so that the pH of the ex vivo biological tissue staining buffer itself automatically decreases from 9.0±0.1 to 7.2-7.8 within 5-7 hours. The C1-C3 short-chain carboxylic acids are one or a combination of the other two of formic acid and acetic acid.

[0016] Specifically, the ex vivo biological tissue staining buffer is placed in a direct current electric field device, the staining molecules and target molecules are added, and the glucose is subjected to a voltage of 20-60 V cm-1 The direct current electric field, the glucose is quantitatively generated C1-C3 short chain carboxylic acid under the driving of the electric field, the ex vivo biological tissue staining buffer itself pH is automatically reduced from 9.0±0.1 to 7.2-7.8 within 5-7 hours, so that the ex vivo biological tissue staining buffer is accurately matched with the optimal binding interval of the antibody, so that the binding ability of the staining molecule and the target molecule is closed-loop controlled.

[0017] More specifically, the glucose as a "programmable acid generator" functions to occur a controllable electrolysis reaction in an electric field environment, release acidic substances such as formic acid / acetic acid in a direct current electric field, dynamically adjust and change the pH value of the buffer system, so as to accurately control the binding ability between the staining molecule and the target molecule, realize the optimization and improvement of the staining effect, and realize the closed-loop control of the automatic electric field-chemical-biological molecule.

[0018] More specifically, further, the clinical human brain, kidney, liver and animal organs (brain, kidney, liver, heart) are pretreated.

[0019] Further, the pretreatment includes tissue fixation, tissue sectioning and tissue transparency.

[0020] Further, the application of the pretreated biological tissue in the staining and imaging in the pH dynamically regulated electric field buffer realizes the detection results of the immunostaining and three-dimensional imaging of the ex vivo human brain, kidney, liver or complete animal organs, and can achieve a staining depth of centimeter level, negligible tissue swelling rate and complete fine structure.

[0021] Further, the biological tissue includes clinical human brain, kidney, liver samples and animal organs.

[0022] Compared with the prior art, the beneficial effects of the present application are:

[0023] 1. The buffer reagent component of the present application based on the electric field-chemical closed-loop pH dynamic regulation is simple, widely applicable, and the buffer pH can be flexibly adjusted according to the characteristics of different staining molecules.

[0024] 2. The buffer reagent proposed in the present application can control the pH range of the buffer system through the oxidation electrolysis of glucose, improve the penetration efficiency and binding ability of the target molecule.

[0025] 3. This invention introduces the concept of "programmable acid generator" into electric field staining for the first time, achieving a triple breakthrough of "deep and uniform, rapid and non-destructive, and low cost", providing an irreplaceable technical solution for high-throughput three-dimensional pathology, whole organ atlas and accurate diagnosis.

[0026] 4. The reagents of this invention have simple components and are universally applicable to various staining molecules such as antibodies and fluorescent dyes, providing an electric field staining technology solution that is efficient, mild and controllable for high-throughput three-dimensional pathology, whole organ mapping and accurate diagnosis.

[0027] 5. Compared with the traditional alkaline borate / Tris system, this invention solves the bottlenecks such as antibody denaturation and deep signal exponential decay caused by "electric field-induced alkaline peaks". It enables human brain, kidney, liver and complete animal organs with centimeter-thickness to obtain high signal-to-noise ratio and uniform thickness immunostaining within 24 hours, and the tissue expansion rate is negligible and the microstructure is completely preserved. Attached Figure Description

[0028] Figure 1 This is a graph showing the pH trend of the buffer solution as a function of electrolysis time in Example 1, Comparative Example 1, and Comparative Example 2 (n=3).

[0029] Figure 2 These are Anti-NeuN signal imaging images of mouse brain tissue samples under different conditions of a novel electric field buffer, with a scale bar of -200 μm. (a), (b), and (c) represent fluorescence images of mouse brain tissue from Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0030] Figure 3 They are from respectively Figure 2 Signal imaging images at different locations under three conditions, where (a), (b), and (c) represent signal imaging images at different depths of the fluorescence images in Example 1, Comparative Example 1, and Comparative Example 2, respectively;

[0031] Figure 4 These are the pH trends of the buffer solutions as a function of electrolysis time in Examples 2, 3, and 4 (n=3).

[0032] Figure 5 These are Anti-Podxl signal imaging images of human brain tissue samples under different conditions of a novel electric field buffer, with a scale bar of -100 μm. (a), (b), and (c) represent fluorescence images of human brain tissue from Example 2, Comparative Example 3, and Comparative Example 4, respectively.

[0033] Figure 6 They are from respectively Figure 5Signal imaging diagrams at different positions under three conditions, wherein (a), (b), (c) represent signal imaging diagrams at different depths of the fluorescence images in Example 2, Comparative Example 3 and Comparative Example 4, respectively;

[0034] Figure 7 is a trend chart of the pH of the buffer in Example 3 varying with electrolysis time (n = 3);

[0035] Figure 8 is a trend chart of the pH of the buffer in Example 4 varying with electrolysis time (n = 3);

[0036] Figure 9 is a comparison diagram of dyeing effects under a direct current electric field and an alternating current electric field in Comparative Example 5, wherein (a): direct current electric field; (b): alternating current electric field, scale bar-100 μm. DETAILED DESCRIPTION

[0037] The present application will be described in detail below with specific implementation cases, which will help those skilled in the art to further understand the present application, but do not limit the present application in any form.

[0038] The purpose of the present application is to provide an ex vivo biological tissue dyeing buffer based on electric field-chemical closed loop pH dynamic regulation, which can maintain a specific pH range of the solution according to the needs during the dyeing process by optimizing the reagent components; under the action of a direct current electric field, its active ingredient glucose acts as a "programmable acid generator" to dynamically adjust the pH value through electrolysis reaction, thereby accurately controlling the binding state of the dyeing molecules and the target molecules. This dynamic regulation mechanism significantly improves the penetration efficiency of the dyeing molecules and the binding ability with the target molecules, so that centimeter-level biological tissues and even complete animal organs can achieve high signal-to-noise ratio, uniform and consistent dyeing effect within 24 hours, greatly improving the dyeing quality and efficiency.

[0039] The buffer system based on the electric field-chemical closed loop of the present application comprises a buffer, a surfactant and a pH regulator, and the proportions are 10-20 mM, 0.05%-0.5%, and 30-50 mM, respectively. Preferably, the concentration of Tris is 20 mM, and the concentration of CAPs is 20 mM; preferably, the concentration of TritonX-100 is 0.2%, and the concentration of Tween20 is 0.5%; preferably, the concentration of glucose is 50 mM. The reagent components can be selected according to the types of tissues, the thickness of tissues and the dyeing molecules, etc.

[0040] Among them, the reagents involved in the present application are commercially available, without special restrictions.

[0041] The biological tissue is from an ex vivo human clinical sample and a mouse organ, including a mouse brain, a kidney, a liver, a heart, a pancreas, and an intestine, but is not limited to the above.

[0042] In the present application, the biological tissue is preferably obtained by heart perfusion, and the animal sample is sequentially perfused with 1xPBS and 4% PFA solution. After the perfusion is completed, the brain is removed by craniotomy and placed in 4% PFA solution pre-cooled at 4°C, and stored in a refrigerator at 4°C overnight.

[0043] In the present application, the biological tissue is preferably pretreated before being dyed with the pH dynamic control electric field buffer; the pretreatment includes tissue sectioning and tissue transparency.

[0044] The biological tissue is dyed with the pH controllable electric field buffer, specifically: for a 2000-micron-thick mouse brain section, the dyeing time of the first and second antibodies is 6 hours; for a 1000-micron-thick human brain section, the dyeing time of the first and second antibodies is 6 hours.

[0045] The imaging of the present application includes but is not limited to confocal microscopy, light sheet microscopy, two-photon microscopy, and wide-field microscopy.

[0046] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0047] The steps of the embodiments of the present application are as follows:

[0048] Embodiment 1: Obtaining a mouse brain section and dyeing it with a pH control electric field

[0049] All animal tests involved in the present application are carried out in accordance with the guidelines of the Animal Ethics Committee of Zhejiang University.

[0050] After the mouse brain tissue is pretreated, it is placed in a device containing an electric field dyeing buffer; the components of the buffer are 20 mM Tris, 0.5% (v / v) Tween20 and 50 mM glucose dissolved in deionized water, and contain Anti-NeuN first antibody diluted at a ratio of 1:200. The direct current electric field dyeing device is turned on, the device voltage is 40V, the initial buffer pH is measured to be 9.0, and is tested every 1 hour, repeated three times, and the pH change is recorded as Figure 1Green marker (a). After 6 hours, the DC field device was turned off and the section was washed in PBS for a total of 3 times, 1.5 hours each. The washed section was then placed back into the DC field device with fresh buffer for the secondary antibody labeling, following the same procedure as above.

[0051] Comparative Example 1: Obtaining a mouse brain section and performing pH regulated field staining

[0052] After pre-treatment, the mouse brain tissue was placed into the device with the field staining buffer; the buffer was composed of 20 mM Tris, 0.5% (v / v) Tween20 and 50 mM glucose dissolved in deionized water, and contained 1:200 diluted Anti-NeuN primary antibody. The DC field staining device was not turned on, and the initial buffer pH was measured to be 9.0. The pH was tested every 1 hour for a total of 3 times, and the pH change was recorded as Figure 1 Red marker (b). After 6 hours, the DC field device was turned off and the section was washed in PBS for a total of 3 times, 1.5 hours each. The washed section was then placed back into the DC field device with fresh buffer for the secondary antibody labeling, following the same procedure as above.

[0053] Comparative Example 2: Obtaining a mouse brain section and performing pH regulated staining

[0054] After pre-treatment, the mouse brain tissue was placed into the device with the field staining buffer; the buffer was composed of 20 mM Tris, 0.5% (v / v) Tween20 and 50 mM glucose dissolved in deionized water, and contained 1:200 diluted Anti-NeuN primary antibody. The DC field staining device was not turned on, and the initial buffer pH was measured to be 9.0. The pH was tested every 1 hour for a total of 3 times, and the pH change was recorded as Figure 1 Blue marker (c). After 6 hours, the section was washed in PBS for a total of 3 times, 1.5 hours each. The washed section was then placed back into the DC field device with fresh buffer for the secondary antibody labeling, following the same procedure as above.

[0055] After the field staining, the mouse brain tissue was transferred to a refractive index matching solution, which was CUBIC-R (antipyrine, nicotinamide and N-butyl diethanolamine dissolved in water with a mass volume ratio of 45%, 30% and 0.5%, respectively) in this case. After the mouse brain tissue was completely transparent, the three groups of transparent mouse brain tissues were imaged using a confocal microscope, and the obtained fluorescence images of the mouse brain tissues in Example 1, Comparative Example 1 and Comparative Example 2 were as follows: Figure 2As shown in (a), (b), and (c). Furthermore, images at different depths were cropped from the three sets of fluorescence images, and the signal image results of the mouse brain tissue fluorescence images at different depths in Example 1, Comparative Example 1, and Comparative Example 2 are as follows. Figure 3 As shown in (a), (b), and (c).

[0056] like Figure 1 As shown, the pH of the buffer solution in group (a) decreased from approximately 9.0 to about 7.3 under a DC electric field. In a DC electric field environment, the aldehyde group of glucose in the alkaline environment is oxidized to a carboxyl group, generating gluconic acid. Under voltage, gluconic acid can further break down to form acidic substances such as formic acid or acetic acid, thus lowering the pH of the buffer solution. In group (b), the pH of the buffer solution without glucose only decreased to about 8.2, and in group (c), the pH of the buffer solution without the DC electric field device remained almost unchanged. The staining effects of the three groups are as follows: Figure 2 As shown in (a), (b), and (c), the staining effects at different depths are respectively as follows: Figure 3 As shown in (a), (b), and (c), the staining effect is the best in the group with the largest pH change, whether considering the overall staining effect or the staining effect at different depths. In the group without glucose, the staining effect becomes worse as you go deeper. The group without the DC electric field device is a normal passive staining, and due to the depth of penetration, most of the signal exists only on the tissue surface.

[0057] Example 2: Obtaining human brain slices and performing pH-controlled electric field staining.

[0058] The 1000-micrometer-thick clinical human brain tissue used in the experiments was obtained from the Chinese Brain Bank of Zhejiang University School of Medicine. After pretreatment (tissue sections, tissue cleared), the human brain tissue sections were placed in a device containing electric field staining buffer. The buffer consisted of 20 mM Tris, 0.2% (v / v) Triton X-100, and 50 mM glucose, and contained a 1:200 dilution of Anti-Podocalyxin primary antibody. The DC electric field staining device was turned on at 40V. The initial pH of the buffer was measured to be 9.0, and the pH was measured every hour for three consecutive times. The pH changes were recorded as follows: Figure 4 As shown in green (a). After 6 hours, the DC electric field device was turned off, and the human brain tissue was washed with PBS three times, each time for 1.5 hours. Afterward, the washed tissue was placed back into the DC electric field device with the buffer solution updated for labeling with the second antibody, following the same procedure as above.

[0059] Comparative Example 3: Obtaining human brain slices and staining them with a pH-controlled electric field.

[0060] The 1000-micron-thick clinical human brain tissue used for the experiment was obtained from the China Brain Bank of Zhejiang University Medical School. After the pretreatment of the human brain tissue slice (tissue slice, tissue transparency), it was placed in the device containing the electric field staining buffer, the composition of the buffer was 20 mM Tris, 0.2% (v / v) Triton X-100 and 0 mM glucose, and contained 1:200 dilution of Anti-Podocalyxin primary antibody, the direct current electric field staining device was turned on, the device voltage was 40 V, the initial pH of the buffer was measured to be 9.0, and was tested every 1 hour for three times, and the pH change was recorded as Figure 4 shown by the red label (b). After 6 hours, the direct current electric field device was turned off, and the human brain tissue was washed with PBS for a total of 3 times, 1.5 hours each time. Then the washed tissue was placed in the direct current electric field device with updated buffer for the labeling of the secondary antibody, and the operation steps were the same as above.

[0061] Comparative Example 4: Obtaining human brain slices and pH-regulated staining thereof

[0062] The 1000-micron-thick clinical human brain tissue used for the experiment was obtained from the China Brain Bank of Zhejiang University Medical School. After the pretreatment of the human brain tissue slice (tissue slice, tissue transparency), it was placed in the device containing the electric field staining buffer, the composition of the buffer was 20 mM Tris, 0.2% (v / v) Triton X-100 and 0 mM glucose, and contained 1:200 dilution of Anti-Podocalyxin primary antibody, the direct current electric field staining device was turned on, the device voltage was 40 V, the initial pH of the buffer was measured to be 9.0, and was tested every 1 hour for three times, and the pH change was recorded as Figure 4 shown by the blue label (c). After 6 hours, the human brain tissue was washed with PBS for a total of 3 times, 1.5 hours each time. Then the washed tissue was placed in the direct current electric field device with updated buffer for the labeling of the secondary antibody, and the operation steps were the same as above.

[0063] After the electric field staining was completed, the human brain tissue was transferred to the refractive index matching liquid, and the refractive index matching liquid used here was CUBIC-R (antipyrine, nicotinamide and N-butyl diethanolamine dissolved in water with a mass-volume ratio of 45%, 30% and 0.5% respectively). After the human brain tissue was completely transparent, a confocal microscope was used to image the three groups of transparent human brain tissue, and the obtained fluorescence images of the human brain tissue in Example 2, Comparative Example 3 and Comparative Example 4 were as shown in (a), (b) and (c) of Figure 5 respectively. And the signal images of different depths of the fluorescence images of the three groups of human brain tissue were obtained as shown in (a), (b) and (c) of Figure 6 respectively.

[0064] As shown in Figure 4 , the pH of the buffer solution in group (a) decreased from the initial 9.0 to about 7.6 under the condition of a direct current electric field. In the alkaline environment under the direct current electric field, the aldehyde group of glucose was oxidized to a carboxyl group to form gluconic acid. Under the voltage, the gluconic acid could be further broken down to generate acidic substances such as formic acid or acetic acid, thereby reducing the pH of the buffer solution. The pH of the buffer solution in group (b) without glucose decreased to about 8.4, and the pH of the buffer solution in group (c) without opening the direct current electric field device hardly changed, further proving that glucose can be electrolyzed into acidic substances under the condition of a direct current electric field, reducing the pH of the buffer solution to improve the binding ability of the antibody and enhance the staining effect. The staining effects of the three groups are shown in Figure 5 (a), (b), and (c), respectively. The staining effects of different depth layers are shown in Figure 6 (a), (b), and (c), respectively. The results are similar to those of the mouse brain. From the overall or different depth staining effects, the staining effect of the group with the largest pH change is the best, while there is almost no signal in the middle position in the group without glucose, and the group without opening the direct current electric field device is normally passively stained. Due to the penetration speed, the signal also only exists on the surface of the tissue. Further proof that the pH change caused by the electrolysis of glucose under the condition of a direct current electric field greatly improves the staining effect of different biological tissues. Example 3

[0065] To further test whether the presence of glucose under the condition of a direct current electric field will gradually reduce the pH of the buffer solution. Use 20 mM CAPs, 0.5% Tween20 and 50 mM glucose buffer solution into the direct current electric field device, open the device voltage is 40V, the initial buffer solution pH is measured to be 9.0, and test every 1 hour, repeat three times, record the pH change as shown in Figure 7 green label (a). After 6 hours, turn off the direct current electric field device. Use 20 mM CAPs, 0.5% Tween20 and 0 mM glucose buffer solution into the direct current electric field device, repeat the above operation, record the pH change as shown in Figure 7 red label (b). Use 20 mM CAPs, 0.5% Tween20 and 50 mM glucose buffer solution into the direct current electric field device, do not open the voltage device, repeat the above operation, record the pH change as shown in Figure 7 blue label (c). Example 4

[0066] The initial pH of the buffer was measured to be 9.0 and was tested every 1 hour for three times. The results were recorded as shown in Figure 8 Figure 1 (a). After 6 hours, the DC electric field device was turned off. The same procedure was repeated using 20 mM CAPs, 0.2% Triton X-100 and 0 mM glucose buffer. The results were recorded as shown in Figure 8 Figure 1 (b). Figure 8 Figure 1 (c).

[0067] As shown in Figure 7 , Figure 8 , the pH of the buffer decreased most when glucose was included and the DC electric field device was turned on. The pH of the buffer decreased less when glucose was not included and the pH of the buffer hardly changed when the DC electric field device was not turned on. This again proved that glucose was electrolyzed to produce acidic substances in the DC electric field environment, which decreased the pH of the buffer and changed the binding ability of the antibody, thus improving the staining results.

[0068] Comparative Example 5:

[0069] The mouse brain tissue was pre-treated and then placed in the electric field staining device containing the buffer. The buffer was composed of 20 mM Tris, 0.5% (v / v) Tween 20 and 50 mM glucose dissolved in deionized water, and contained Anti-TH primary antibody diluted at a ratio of 1:200. The AC electric field staining device was turned on and the voltage of the device was 40 V. After 6 hours, the electric field device was turned off. The mouse brain tissue was washed and labeled with the secondary antibody according to the above procedure. The results were imaged as shown in Figure 9 Figure 2 (a).

[0070] The mouse brain tissue was pre-treated and then placed in the electric field staining device containing the buffer. The buffer was composed of 20 mM Tris, 0.5% (v / v) Tween 20 and 50 mM glucose dissolved in deionized water, and contained Anti-TH primary antibody diluted at a ratio of 1:200. The AC electric field staining device was turned on and the voltage of the device was 40 V. After 6 hours, the electric field device was turned off. The mouse brain tissue was washed and labeled with the secondary antibody according to the above procedure. The results were imaged as shown in Figure 9 Figure 2 (b).

[0071] Because the size and polarity of the direct current voltage is always constant, the dyeing molecules are affected by the "constant electrostatic force" in the electric field, and this "one-way, uninterrupted" migration helps the dyeing molecules to quickly and uniformly dye, while the size and polarity of the alternating current voltage will periodically alternate, causing the dyeing molecules to always do "reciprocating motion" in the dyeing solution, and unable to form a "continuous migration to the tissue" movement trend, greatly affecting the dyeing effect.

[0072] In summary, the application first introduces the "electric field-glucose coupled cascade acid generation" mechanism into the dyeing buffer system, and truly realizes the "on-site programming and regulation" of pH. In the 20-60 V cm -1 In the direct current electric field, glucose is directionally oxidized into formic acid / acetic acid and other substances, so that the pH of the system is linearly reduced from 9.0 to 7.2-7.8. The closed-loop regulation breaks through the long-standing "alkaline barrier" problem of traditional electric field dyeing, reduces the immunolabeling time of centimeter-level tissues from 15 days to 24 hours or less, significantly improves the signal uniformity, signal-to-noise ratio and other indicators, and the tissue swelling rate is negligible, and the microstructure is not damaged. Compared with the existing electric field buffer, the composition of the present scheme is extremely simple, the cost is extremely low, and the universality is extremely high, which provides a technical route with high efficiency and economy for deep, fast and non-destructive biological tissue dyeing, and has outstanding substantial features and significant progress.

[0073] Those skilled in the art will readily understand that the above description is only a preferred embodiment of the present application, and is not intended to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An ex vivo biological tissue staining buffer based on electric field-chemical closed-loop pH dynamic regulation, characterized in that, The ex vivo biological tissue staining buffer is formed mainly by a buffer, a surfactant and a pH regulator dissolved in deionized water, wherein the pH regulator is glucose, and the ex vivo biological tissue staining buffer is placed in a direct current electric field device; The initial pH value of the ex vivo biological tissue staining buffer is set to 9.0±0.

1. The molar concentration of the pH regulator in the ex vivo biological tissue staining buffer is 30-50 mM.

2. The ex vivo biological tissue staining buffer based on the electrochemical closed-loop pH dynamic regulation according to claim 1, characterized in that, The molar concentration of the buffer in the ex vivo biological tissue staining buffer is 10-20 mM, and the buffer is one or a combination of the other of tris(hydroxymethyl)aminomethane and 3-(cyclohexylamino)-1-propanesulfonic acid.

3. The ex vivo biological tissue staining buffer based on the electrochemical closed-loop pH dynamic regulation of claim 1, wherein, The volume concentration of the surfactant in the ex vivo biological tissue staining buffer is 0.05-0.5% v / v, and the surfactant is one or a combination of Triton X-100 and Tween 20.

4. The use of the ex vivo biological tissue staining buffer based on the electric field-chemical closed-loop dynamic pH regulation according to any one of claims 1-3, characterized in that, Immunostaining detection and three-dimensional imaging of ex vivo biological tissues. 5.The application of the ex vivo biological tissue staining buffer based on the electric field-chemical closed-loop pH dynamic regulation according to claim 4, characterized in that, The ex vivo biological tissue comprises an ex vivo human brain, kidney, liver or complete animal organ, and the results obtained by three-dimensional imaging of the ex vivo biological tissue in the ex vivo biological tissue staining buffer include one or a combination of staining depth, tissue swelling rate and fine structure of the ex vivo biological tissue.

6. The method for dynamic pH regulation by electro-chemical closed loop according to any one of claims 1-3, wherein the buffer solution for staining biological tissues ex vivo is applied. The ex vivo biological tissue staining buffer is placed in a direct current electric field device, and the direct current electric field device is turned on, wherein the glucose generates C1-C3 carboxylic acid under the driving of the direct current electric field, so that the pH of the ex vivo biological tissue staining buffer itself decreases within 5-7 hours.

7. The electro-chemical closed-loop pH dynamic regulation method of claim 6, wherein, The initial pH value of the ex vivo biological tissue staining buffer is set to 9.0±0.

1. 8.The electric field-chemical closed-loop dynamic pH regulation method of claim 6, wherein, The glucose in the ex vivo biological tissue staining buffer generates C1-C3 carboxylic acid under the driving of the direct current electric field, so that the pH of the ex vivo biological tissue staining buffer itself automatically decreases from 9.0±0.1 to 7.2-7.8 within 5-7 hours. 9.The electric field-chemical closed-loop pH dynamic regulation method of claim 6, wherein, The C1-C3 carboxylic acid is one or a combination of formic acid and acetic acid.

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