Precise immunofluorescence labeling method for integral tissue sample

A tissue sample and immunofluorescence technology, applied in the biological field, can solve the problems of low image resolution, poor compatibility of fluorescent proteins, and low permeability of optical clearing agents, etc., to achieve clear neurons and blood vessels, and bright fluorescent markers.

Pending Publication Date: 2021-05-25
佳维斯(武汉)生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the physical tomography technology of the prior art has low permeability of optically transparent agents, and poor compatibility of fluorescent proteins leads to low image resolution and cannot display complete fluorescence images of tissues.

Method used

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  • Precise immunofluorescence labeling method for integral tissue sample
  • Precise immunofluorescence labeling method for integral tissue sample
  • Precise immunofluorescence labeling method for integral tissue sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Sample Collection:

[0041] S1, first anesthetize the animal, and select the intraperitoneal injection of pentobarbital to anesthetize the mouse;

[0042] S2, the heart of the anesthetized mouse was perfused with PBS and 4% PFA sequentially;

[0043] S3, mice were dissected after perfusion, and mouse embryonic tissues were isolated;

[0044] S4, fix the embryo tissue with 4% PFA at 4°C, shake overnight, and then fix at 20-30°C for 1 hour;

[0045] S5, the embryo tissue was washed with PBS buffer at room temperature for 3 times, 30 minutes each time, and then the embryo tissue was placed in PBS buffer and stored at 4°C.

[0046] The fluorescent labeling method is as follows:

[0047] Transparent agent: the pH value is 7.5, including 2% urea by mass fraction, 20% DMSO by volume fraction, 2% sucrose by mass fraction, 3% SDS by mass fraction, 1% glutathione by mass fraction and 0.02% sodium azide and PBS buffer.

[0048] Blocking solution: PBS buffer, tritonx-100 and s...

Embodiment 2

[0064] Sample Collection:

[0065] S1, first anesthetize the animal, and select the intraperitoneal injection of pentobarbital to anesthetize the mouse;

[0066] S2, the heart of the anesthetized mouse was perfused with PBS and 4% PFA sequentially;

[0067] S3, the mouse was dissected after perfusion, and the brain tissue of the mouse was isolated;

[0068] S4, fix the brain tissue with 4% PFA at 4°C, shake overnight, and then fix at 20-30°C for 1 hour;

[0069] S5, the brain tissue was washed with PBS buffer solution at room temperature for 3 times, 30 minutes each time, and then the brain tissue was placed in PBS buffer solution and stored at 4°C.

[0070] The fluorescent labeling method is as follows:

[0071] Transparent agent: the pH value is 8.5, including the urea of ​​3% by mass fraction, the DMSO of 30% by volume fraction, the sucrose of 5% by mass fraction, the SDS of 5% by mass fraction, the glutathione and the glutathione of 3% by mass fraction 0.05% sodium azi...

Embodiment 3

[0087] Sample collection:

[0088] S1, first anesthetize the animal, and select the intraperitoneal injection of pentobarbital to anesthetize the mouse;

[0089] S2, the heart of the anesthetized mouse was perfused with PBS and 4% PFA sequentially;

[0090] S3, the mouse was dissected after perfusion, and the brain tissue of the mouse was isolated;

[0091] S4, fix the brain tissue with 4% PFA at 4°C, shake overnight, and then fix at 20-30°C for 1 hour;

[0092] S5, the brain tissue was washed with PBS buffer solution at room temperature for 3 times, 30 minutes each time, and then the brain tissue was placed in PBS buffer solution and stored at 4°C.

[0093] The fluorescent labeling method is as follows:

[0094] Transparent agent: the pH value is 8, including the urea of ​​2.5% by mass fraction, the DMSO of 25% by volume fraction, the sucrose of 3% by mass fraction, the SDS of 4% by mass fraction, the glutathione of 2% by mass fraction and the mass fraction of 0.03% sodiu...

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Abstract

The invention provides a precise immunofluorescence labeling method for a whole tissue sample, which comprises the following steps: fixing, pretreatment, transparentizing treatment, confining liquid confining, freeze thawing and primary antibody incubation, and secondary antibody incubation. Freeze thawing and ultrasonic incubation are adopted, so that the permeability of an optical transparentizing agent and the compatibility of fluorescent protein are increased, the resolution of a fluorescence image is increased, the permeability of a large tissue sample is realized, and the overall immunolabeling of an organ is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a precise immunofluorescent labeling method for whole tissue samples. Background technique [0002] Immunofluorescence technology utilizes the principle of highly specific combination of antigen and antibody, couples a measurable substance to the antibody or antigen, and qualitatively detects the antigen or antibody in the sample by detecting the presence or absence and content of the marker. , positioning and quantitative detection. Especially in the past 30 years, immunofluorescence technology is the fastest-growing and most widely used technology in labeling immunology technology, which has the advantages of strong specificity, high sensitivity, fast detection speed and easy observation. [0003] For optical imaging of biological tissues labeled with fluorescent probes, there are mainly two existing physical tomography techniques: one is optical tomography, which optically suppre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533
CPCG01N33/533
Inventor 姚应涛田天田根根
Owner 佳维斯(武汉)生物医药有限公司
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