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Weak background fluorescence type resin embedding method

A technology of resin package and fluorescence, which is applied in the field of resin embedding with weak background fluorescence, can solve the problems of quenching and XFPs fluorescence intensity reduction, and achieve the effect of low cost, simple operation steps, and background fluorescence reduction

Active Publication Date: 2014-05-21
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, some extreme physical and chemical conditions, such as high temperature, strong redox agent, strong acid and strong alkali, etc., will lead to the reduction or even quenching of the fluorescence intensity of XFPs.

Method used

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  • Weak background fluorescence type resin embedding method
  • Weak background fluorescence type resin embedding method

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Effect test

Embodiment 1

[0027] This embodiment provides a resin embedding method with weak background fluorescence, comprising the following steps:

[0028] (1) Adult Thy1-GFP-M line transgenic mice were anesthetized with 1% pentobarbital sodium, and perfused with 0.01mol / L PBS solution at 37°C for 3 minutes through the left ventricle of the heart. After the blood was washed clean, the perfusion was performed immediately Pre-cooled 4% paraformaldehyde fixative in ice water for 1 hr. Then, the whole brain of the mouse was taken out and put into ice water pre-cooled 4% paraformaldehyde fixative again and fixed for 24 hours. 2.5% sucrose was added to the fixative, and the solvent was 0.01mol / L PBS.

[0029] (2) After fixation, rinse the whole brain sample in 0.01mol / L PBS pre-cooled in ice water for 24 hours, during which time the new solution was replaced 5 times to thoroughly wash away the residual paraformaldehyde fixative solution.

[0030] (3) Next, the whole brain samples were sequentially place...

Embodiment 2

[0035] This embodiment provides a resin embedding method with weak background fluorescence, comprising the following steps:

[0036] (1) Adult Thy1-GFP-M line transgenic mice were anesthetized with 1% pentobarbital sodium, and perfused with 0.01mol / L PBS solution at 37°C for 3 minutes through the left ventricle of the heart. After the blood was washed clean, the perfusion was performed immediately Pre-cooled 4% paraformaldehyde fixative in ice water for 1 hr. Then, the whole brain of the mouse was taken out, cut into 1 mm thick brain blocks, and put into 4% paraformaldehyde fixative pre-cooled in ice water again and fixed for 6 hours. 2.5% sucrose was added to the fixative, and the solvent was 0.01mol / L PBS.

[0037] (2) After the fixation, the brain block samples were rinsed in 0.01mol / L PBS pre-cooled in ice water for 3 hours, during which the new solution was replaced 3 times to thoroughly wash away the residual paraformaldehyde fixative solution.

[0038] (3) Next, put t...

Embodiment 3

[0042] This embodiment provides a resin embedding method with weak background fluorescence, comprising the following steps:

[0043] (1) Adult Thy1-YFP-H line transgenic mice were anesthetized with 1% pentobarbital sodium, and perfused with 0.01mol / L PBS solution at 37°C for 3 minutes through the left ventricle of the heart. After the blood was washed clean, the perfusion was performed immediately Pre-cooled 4% paraformaldehyde fixative in ice water for 1 hr. Then, the whole brain of the mouse was taken out, cut into 5 mm brain blocks, and put into 4% paraformaldehyde fixative pre-cooled in ice water again and fixed for 12 hours. 2.5% sucrose was added to the fixative, and the solvent was 0.01mol / LPBS.

[0044] (2) After the fixation, the brain block samples were rinsed in 0.01mol / L PBS pre-cooled in ice water for 12 hours, during which the new solution was replaced 4 times to thoroughly wash away the residual paraformaldehyde fixative solution.

[0045] (3) Next, put the wh...

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Abstract

The invention discloses a weak background fluorescence type resin embedding method and belongs to the technical field of biological engineering. In the method, a fluorescence biological sample is permeated and embedded by using a resin solution in which Sudan black B is dissolved as a dying and embedding solution. On the premise that a biological sample obtained by adopting the method keeps the fluorescence signal intensity, the background fluorescence is greatly reduced. The method can be applicable to a large sample with a centimeter size; the obtained biological sample can be used for continuously slicing in an ultrathin manner. The method provided by the invention has the advantages of being low in cost and simple in operation steps, and is applicable to popularization and application in ordinary laboratories.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a resin embedding method with weak background fluorescence. Background technique [0002] The grand challenge of modern biology is to understand the structure and function that spans the spectrum from cells to tissues to organs and even to complete organisms. Due to the influence of depth of field and background fluorescence, traditional epi-fluorescence microscopy imaging can only image thin sections of tens of microns. Combining this block-surface imaging method with sequential mechanical thin sections enables successful rapid 3D imaging of centimeter-scale large-scale biological samples. Usually, in order to ensure that biological samples can be sliced ​​serially, they need to be embedded with resin. However, although this technology solves the problem of imaging depth of field, it still has the problem of background fluorescence interference. Large-sized biological ...

Claims

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Application Information

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IPC IPC(8): G01N1/36G01N1/30
Inventor 龚辉骆清铭胡碧荷
Owner HUAZHONG UNIV OF SCI & TECH
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