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Resin embedding method for biological tissues marked by fluorescent protein and application of alkaline solution

A technology of biological tissue and fluorescent protein, which is applied in the application of alkaline solution, and in the field of resin embedding of fluorescent protein-labeled biological tissue, can solve the problems of unavoidable fluorescence quenching, low fluorescence brightness, poor imaging effect, etc., and achieve fluorescence intensity And the effects of good image quality, improved fluorescence intensity, and excellent cutting performance

Inactive Publication Date: 2015-11-04
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot avoid the fluorescence quenching phenomenon, the fluorescence brightness is low, and the imaging effect is poor.
[0004] The current resin embedding technology, the phenomenon of fluorescence quenching is common, and the imaging effect is not ideal, especially for large biological samples
Moreover, due to the lack of understanding of the mechanism of fluorescent protein quenching during resin embedding, the phenomenon of fluorescence quenching is uncontrollable.
Both physical and chemical fixation embedding techniques are empirical, their portability is severely limited, and one approach is often only applicable to specific tissues and fluorescent proteins

Method used

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  • Resin embedding method for biological tissues marked by fluorescent protein and application of alkaline solution
  • Resin embedding method for biological tissues marked by fluorescent protein and application of alkaline solution
  • Resin embedding method for biological tissues marked by fluorescent protein and application of alkaline solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The resin embedding method of the whole brain of thy1-YFPH mice includes the following steps:

[0053] (1) The sample is fixed.

[0054] The GFP derivative, enhanced yellow fluorescent protein (EYFP)-labeled mouse whole brain is fixed by chemical fixation means to obtain fixed EYFP-labeled biological tissue.

[0055] Specific steps are as follows:

[0056] After the heart was perfused at 4°C, the dissected whole brain of the mouse was soaked in 4% PFA solution for about 12 hours. Use 40ml of PBS solution, rinse for four hours each time.

[0057] (2) Sample dehydration.

[0058] The fixed EYFP-labeled mouse whole brain was replaced with ethanol, so that the biological tissue was dehydrated, and the dehydrated EYFP-labeled mouse whole brain was obtained.

[0059] The specific steps are:

[0060] At 4°C, the fixed EYFP-labeled mouse whole brain was sequentially passed through 20 ml of gradient ethanol double-distilled water solution, soaked for 2 hours, and dehydrated...

Embodiment 2

[0073] The resin embedding method of the whole brain of thy1-GFPM mice comprises the following steps:

[0074] (1) The sample is fixed.

[0075] The GFP derivative, enhanced green fluorescent protein (EGFP)-labeled mouse whole brain is fixed by chemical fixation means to obtain fixed EGFP-labeled biological tissue.

[0076] Specific steps are as follows:

[0077] After perfusing the heart at 4°C, the dissected whole brain of the mouse was soaked in formalin solution for about 12 hours. The amount of formalin solution was 20ml per mouse, and then rinsed with PBS solution three times, each Use 40ml of PBS solution, rinse for four hours each time.

[0078] (2) Sample dehydration.

[0079] The fixed EGFP-labeled mouse whole brain was replaced with ethanol, so that the biological tissue was dehydrated, and the dehydrated EGFP-labeled mouse whole brain was obtained.

[0080] The specific steps are:

[0081] At 4°C, the fixed EGFP-labeled mouse whole brain was sequentially passe...

Embodiment 3

[0094] GFP-labeled mouse brain resin embedding method, comprising the following steps:

[0095] (1) The sample is fixed.

[0096] The green fluorescent protein (GFP)-labeled mouse whole brain was fixed by chemical fixation means to obtain fixed GFP-labeled biological tissue.

[0097] Specific steps are as follows:

[0098] After the heart was perfused at 4°C, the dissected whole brain of the mouse was soaked in 4% PFA solution for about 12 hours. Use 40ml of PBS solution, rinse for four hours each time.

[0099] (2) Sample dehydration.

[0100] The fixed GFP-labeled mouse whole brain was replaced with ethanol, so that the biological tissue was dehydrated, and the dehydrated GFP-labeled mouse whole brain was obtained.

[0101] The specific steps are:

[0102]At 4°C, dehydrate the fixed GFP-labeled mouse whole brain through 20 ml of gradient ethanol double distilled water successively for 2 hours. The concentration gradient of ethanol double distilled water solution is 50%...

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Abstract

The invention discloses a resin embedding method for biological tissues marked by fluorescent protein. The resin embedding method comprises the following steps: (1), fixing the biological tissues marked by the fluorescent protein in a chemical fixing manner to obtain the fixed biological tissues marked by the fluorescent protein; (2), replacing the fixed biological tissues marked by the fluorescent protein with an organic solvent, so that the biologic tissues are dewatered to obtain dewatered biological tissues marked by the fluorescent protein; (3), performing embedding agent permeation treatment on the dewatered biological tissues marked by the fluorescent protein to obtain embedding-agent monomer-filled biological tissues marked by the fluorescent protein; (4), enabling the embedding agent to generate a polymerization reaction to obtain a resin-embedding sample of the biological tissues marked by the fluorescent protein; (5), soaking the resin-embedding sample in an alkaline solution, and adjusting the pH value to be 8-12. The resin embedding method for the biological tissues marked by the fluorescent protein can be suitable for biologic tissue samples of various sizes, the fluorescence intensity is high, the imaging effect is good, and the samples have excellent cutting performance.

Description

technical field [0001] The invention belongs to the field of fluorescence microscopic imaging, and more specifically relates to a method for embedding fluorescent protein-labeled biological tissue resin and the application of alkaline solution. Background technique [0002] Resin-embedded samples have superior machinability and are the standard method for preparing ultra-thin tissue sections for electron microscopy as well as samples for electron microscopy or optical tomography microscopy. However, due to the severe quenching of green fluorescent protein and its derivatives during the resin embedding process, dehydration and organic phase treatment, the resin embedding technology cannot be compatible with the fluorescent labeling technology based on modern molecular biology. [0003] There are two main types of existing resin embedding techniques: embedding methods based on physical fixation means, for samples with small volume (thickness not exceeding 0.6 mm), resin encaps...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/36
Inventor 曾绍群龚辉骆清铭熊汗青
Owner HUAZHONG UNIV OF SCI & TECH
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