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Rapid biological tissue labeling method

A technology for labeling and tissue samples, used in instruments, measuring devices, scientific instruments, etc., can solve the problems of sample heating, permanent deformation, uneven antibody labeling, etc.

Active Publication Date: 2021-06-29
李小卫
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current technical methods all use relatively high electric fields (>1000V / m), which will produce obvious boundary effects of antibody aggregation at the tissue interface
The boundary effect will cause the pores on the surface of the tissue to be filled by a large number of antibody molecules, which seriously prevents subsequent antibody molecules from entering the tissue, making the antibody labeling inside and outside the tissue severely uneven, and it is difficult to achieve high-quality immunofluorescence labeling of transparent tissue
At the same time, the high current corresponding to the strong electric field will cause the sample to heat up significantly, and the final sample damage or permanent deformation due to the change of the local electric field and / or the increase of temperature

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Embodiment 1 is carried out the experiment of electric field parameter optimization to brain tissue

[0138] Such as figure 1 , as shown in A, a 500μm brain slice that has been transparentized by CLARITY was sandwiched between two coverslips, both sides were sealed with blue rubber and then fixed with epoxy resin, and 1:1 was added to one side of the brain slice. Anti-GFP 555 antibody solution at a concentration of 200 (diluted in electrophoresis solution), installed in a glass dish. Both sides of the sealant were dammed and fixed with epoxy resin, and an appropriate amount of electrophoretic liquid was added to the electrodes on both sides until the liquid just covered the electrode wire, but did not cover the sealant. Using 0.05mA / mm 2 、0.1mA / mm 2 、0.2mA / mm 2 、 0.3mA / mm 2 、0.4mA / mm 2 and 0.5mA / mm 2 The current density experiments were carried out. The electrophoresis solution was prepared as 0.1M boric acid solution (containing 0.1% Triton X-100, pH=8.6). The...

Embodiment 2

[0141] Example 2 Staining Histone 3 (H3) to 2mm thick brain tissue sections

[0142] The sample is installed as image 3 b, The tissue was embedded in two 8% agarose gel rings, placed in the sample compartment. Insert the sample chamber again image 3 For the antibody chamber in c, after leak detection, add antibody diluent to the two tanks of the antibody chamber. Finally as image 3 a, Insert the antibody chamber into the electrophoresis chamber, and add the electrophoresis solution into the two slots of the electrophoresis chamber. The side sectional view of the final whole device after installation is as follows: image 3 as shown in d.

[0143] Such as Figure 4 A Confocal imaging was performed. The electrophoresis buffer was 0.1M boric acid, pH 8.6 (adjusted with NaOH), containing 0.1% Triton X-100. The antibody diluent was diluted with 5% BSA prepared by the antibody (Histone H3 XP Rabbit mAb, Alexa-647 conjugate, 12230S, Cell Signaling Technology) with electrop...

Embodiment 3

[0144] Example 3 Staining lamin B1 to 2mm thick brain tissue sections

[0145] The sample is installed as image 3 b, The tissue was embedded in two 8% agarose gel rings, placed in the sample compartment. Insert the sample chamber again image 3 For the antibody chamber in c, after leak detection, add antibody diluent to the two tanks of the antibody chamber. Finally as image 3 a, Insert the antibody chamber into the electrophoresis chamber, and add the electrophoresis solution into the two slots of the electrophoresis chamber. The side sectional view of the final whole device after installation is as follows: image 3 as shown in d.

[0146] Such as Figure 4 A Confocal imaging was performed. The electrophoresis buffer was 0.1M boric acid, pH 8.6 (adjusted with NaOH), containing 0.1% Triton X-100. The antibody diluent was diluted with 5% BSA prepared from the primary antibody (Anti-lamin B1 antibody, ab16048, Abcam) with electrophoresis buffer to a concentration of 1...

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Abstract

The invention provides a rapid biological tissue labeling method, and particularly provides a method for performing immunolabeling on a tissue sample, which comprises the following steps: (a) providing a labeling system which contains a tissue sample to be subjected to immunolabeling, a probe for labeling the tissue sample and a buffer solution; and (b) placing the labeling system under the action of an electric field for labeling treatment, so that the probe enters the tissue sample, the tissue sample being subjected to immunolabeling, obtaining the immunolabeled tissue sample, and the electric field having the following characteristics that the change range of current density being 0.1-0.3 mA / mm<2>. According to the method disclosed by the invention, the time of complete and transparent tissue immunolabeling can be remarkably shortened, the macroscopic structure and the microstructure of the tissue cannot be damaged, and the integrity of the tissue is kept.

Description

technical field [0001] The invention relates to the field of biological devices, in particular to a method for rapid biological tissue marking. Background technique [0002] In recent years, advances in biological tissue clearing technology have enabled people to obtain the 3D spatial structure of intact tissues at the single-cell scale directly through fluorescence microscopy. The combination of tissue clearing and the latest imaging technology has completely changed the ability of people to obtain the structure and function information of these complex biological tissues under physiological and pathophysiological conditions, and has revolutionized the research methods of histology and pathology. potential scientific value and clinical application value. [0003] However, new technologies bring new problems, and the routine application of these technologies is often hindered by the lengthy process of immunofluorescence labeling. Because these techniques typically rely on ...

Claims

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Application Information

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IPC IPC(8): G01N33/532G01N33/533
CPCG01N33/532G01N33/533
Inventor 李小卫邵志峰张倪
Owner 李小卫
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