A method for reducing background fluorescence of biological samples

A biological sample and fluorescence technology, which is applied in the field of reducing the background fluorescence of biological samples, can solve the problems of no research on biological tissue blocks, cumbersome experimental procedures, uneven staining, etc., and achieve the effects of short processing time, reduced background fluorescence, and simple operation steps

Active Publication Date: 2017-03-15
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current staining methods are limited to cell solutions or slices of tens of microns, and have not studied millimeter-scale or even centimeter-scale biological tissue blocks.
However, when the sample size increases, it often leads to slow dyeing time, uneven dyeing, and cumbersome experimental procedures.

Method used

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  • A method for reducing background fluorescence of biological samples

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Effect test

Embodiment 1

[0022] This embodiment provides a method for reducing the background fluorescence of biological samples, comprising the following steps:

[0023] The biological sample is a Thy1-eYFP-H mouse brain block with a thickness of 3 mm, and the imaging system is an Olympus IX71 wide-field fluorescence microscope.

[0024] (1) Thy1-eYFP-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-cool 4% paraformaldehyde fixative in ice water for 1 hour. Then, the whole brain of the mouse was taken out, cut into 3 mm thick 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 PBS with a concentration of 0.01mol / L.

[0025] (2) After the fixation, the whole brain samples were rinsed...

Embodiment 2

[0030] This embodiment provides a method for reducing the background fluorescence of biological samples, comprising the following steps:

[0031] The biological sample is a Thy1-eYFP-H mouse whole brain sample.

[0032] (1) Thy1-eYFP-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-cool 4% paraformaldehyde fixative in ice water for 1 hour. 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 PBS with a concentration of 0.01mol / L.

[0033] (2) After the fixation, the whole brain samples were rinsed in PBS with a concentration of 0.01 mol / L pre-cooled in ice water for 24 hours, during which the new solution was replaced 5 times to...

Embodiment 3

[0038] This embodiment provides a method for reducing the background fluorescence of biological samples, comprising the following steps:

[0039] The biological samples were brain blocks of Thy1-eYFP-H mice with a thickness of 1 mm.

[0040] (1) Thy1-eYFP-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-cool 4% paraformaldehyde fixative in ice water for 1 hour. Then, the whole brain of the mouse was taken out, cut into 1 mm thick brain blocks, and put into ice water pre-cooled 4% paraformaldehyde fixative solution again and fixed for 6 hours. 2.5% sucrose was added to the fixative, and the solvent was PBS with a concentration of 0.01mol / L.

[0041] (2) After the fixation, the whole brain samples were rinsed in PBS with a concentration of 0.01mol / L pre-cooled in ice water for...

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Abstract

The invention discloses a method for reducing background fluorescence of a biological sample, and belongs to the technical field of biological engineering. The method is characterized in that sudan black B is used as a light absorbing agent, a high-concentration ethanol solution is used for dissolving the sudan black B dying liquid, so that the sudan black B dying liquid can be rapidly and uniformly permeated into biological tissues. By utilizing the method, the background fluorescence of the biological sample is greatly reduced, and the signal contrast of the fluorescent imaging can be improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for reducing background fluorescence of biological samples. Background technique [0002] Epifluorescence microscopy imaging is one of the most commonly used microscopy techniques in biological research. After combining with fluorescent proteins (GFP and its derivatives, XFPs), immunofluorescence, and fluorescent probe labeling methods, it has been widely used in zoology, plant It has been widely used in the fields of biology, microbiology, immunology, pathology and pharmacy. [0003] However, there is a very serious problem in epi-fluorescence imaging, which is the interference of background fluorescence, especially for thicker biological tissue blocks. Background fluorescence may not only lead to false effective signals, but even make the signal in the focal plane be annihilated. In biological research, not only local fine structures are often required, but al...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28
Inventor 龚辉骆清铭胡碧荷
Owner HUAZHONG UNIV OF SCI & TECH
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