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Plastic embedding method for multicolor fluorescent labeled sample

A fluorescent labeling and sample technology, applied in the field of bioengineering, can solve problems such as limited application and reduction

Inactive Publication Date: 2018-11-16
HUST SUZHOU INST FOR BRAINMATICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing resin embedding method uses pure resin or resin samples dissolved in SBB to infiltrate and embed fluorescent biological tissues to obtain samples with reduced background fluorescence. Its shortcoming is that this embedding method is only suitable for green fluorescent protein Labeled biological tissue is not suitable for other fluorescent (such as red fluorescent protein, various fluorescein probes, etc.) labeling, which greatly limits its application, such as figure 1 shown

Method used

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  • Plastic embedding method for multicolor fluorescent labeled sample
  • Plastic embedding method for multicolor fluorescent labeled sample
  • Plastic embedding method for multicolor fluorescent labeled sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Adult VIP::Ai14 hybrid mice were anesthetized with 1% pentobarbital sodium, and perfused by cardiac perfusion, first perfused with 0.01M PBS solution at 37°C for 3-7 minutes, and then perfused with ice water pre-cooled 4% Paraformaldehyde fixative for 7-10 minutes. After cardiac perfusion, the mouse brain tissue was taken, put into 4% paraformaldehyde fixative solution, and post-fixed at 4°C for 6-24 hours. The 4% paraformaldehyde fixative is prepared by dissolving 4% paraformaldehyde and 2.5% sucrose in 0.01M PBS.

[0040] The post-fixed brain tissue was rinsed in 0.01M PBS pre-cooled with ice water for 12-24 hours, during which the new rinse solution was replaced 3-5 times to thoroughly wash away the fixative in the tissue.

[0041] After rinsing, the brain tissue was pre-cooled in ice water and added to 50%, 70% and 95% ethanol solutions of 0.5%-0.7% DTT in turn, and dehydrated in gradients for 1-2 hours each time.

[0042] After dehydration, put the brain tissue i...

Embodiment 2

[0048] Take tdTomato-labeled mouse heart tissue, put it into 4% paraformaldehyde fixative solution, and post-fix at 4°C for 6-24 hours. The 4% paraformaldehyde fixative is prepared by dissolving 4% paraformaldehyde and 2.5% sucrose in 0.01M PBS.

[0049] The post-fixed heart tissue was rinsed in 0.01M PBS pre-cooled with ice water for 12-24 hours, during which the new rinsing solution was replaced 3-5 times to thoroughly wash away the fixative in the tissue.

[0050] After rinsing, the heart tissue was pre-cooled in ice water and added with 0.5%-0.7% DTT in 50%, 70% and 95% ethanol solutions for gradient dehydration, 0.5-1 hour each time.

[0051]After dehydration, put the heart tissue into 50%, 70%, 85% and 100% GMA solutions containing 0.1%-0.2% SBB and 0.7%-1% DTT in gradient osmosis, each gradient for 1-2 hours , in which, the solvent of 50%, 70% and 85% GMA permeate was 95% ethanol; subsequently, the heart tissue was put into fresh 100% resin solution adding 0.1%-0.2% SB...

Embodiment 3

[0056] The rat spinal cord tissue labeled with red fluorescein was taken, put into 4% paraformaldehyde fixative solution, and post-fixed at 4°C for 6-24 hours. The 4% paraformaldehyde fixative is prepared by dissolving 4% paraformaldehyde and 2.5% sucrose in 0.01M PBS.

[0057] The post-fixed spinal cord tissue was rinsed in ice water pre-cooled 0.01M PBS for 6-12 hours, during which the new rinse solution was replaced 3-5 times to thoroughly wash away the fixative in the tissue.

[0058] After rinsing, put the spinal cord tissue into ice water pre-cooling and adding 0.5%-0.7% DTT in 50%, 70% and 95% ethanol solution successively, and carry out gradient dehydration, 0.5-1 hour each time.

[0059] After dehydration, put the spinal cord tissue into 50%, 70%, 85% and 100% GMA solutions containing 0.1%-0.2% SBB and 0.7%-1% DTT in gradient osmosis, each gradient for 1-2 hours , where the solvent of 50%, 70% and 85% GMA infiltrates is 95% ethanol; subsequently, put the spinal cord ...

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Abstract

The invention discloses a plastic embedding method for a multicolor fluorescent labeled sample. The plastic embedding method comprises the steps as follows: carrying out preprocessing on a biologicalsample; sequentially placing the preprocessed biological sample into resin solutions of SBB and DTT, which has different mass concentrations, to carry out gradient penetration for 0.5 to 3 hours in each concentration of the resin solution, and then placing the biological sample into a fresh resin solution added with SBB and DTT again to continuously carry out penetration for 6 to 12 hours; and carrying out embedding on the biological sample after the gradient penetration by resin penetrating fluid, and heating to obtain the embedded fluorescent labeled sample. The embedding method provided bythe invention is applicable to embedding of various resin, and can be applicable to the multicolor fluorescent labeled sample.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for plastically embedding multicolor fluorescently labeled samples. Background technique [0002] At present, through fluorescent labeling technology, various information can be marked on a sample at the same time, which plays an important role in the in-depth study of the structure and function of organisms. [0003] The existing resin embedding method uses pure resin or resin samples dissolved in SBB to infiltrate and embed fluorescent biological tissues to obtain samples with reduced background fluorescence. Its shortcoming is that this embedding method is only suitable for green fluorescent protein Labeled biological tissue is not suitable for other fluorescent (such as red fluorescent protein, various fluorescein probes, etc.) labeling, which greatly limits its application, such as figure 1 shown. Contents of the invention [0004] In view of this, the pr...

Claims

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Application Information

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IPC IPC(8): G01N1/36G01N1/30
CPCG01N1/30G01N1/36G01N2001/364
Inventor 龚辉李向宁任淼田娇娇
Owner HUST SUZHOU INST FOR BRAINMATICS
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