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An immunofluorescent staining method for synapse sites in a nervous system

A technology of immunofluorescence staining and nervous system, which is applied in the preparation, sampling, and measuring devices of test samples, can solve the problems of Caenorhabditis elegans worms unable to maintain the structure domain shape, and improve the fixation effect and fixation rate. Easy to dye and maintain the integrity of the effect

Active Publication Date: 2016-05-04
武汉百齐生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, many scientists have begun to use Caenorhabditis elegans for application development research, but when using C. elegans for immunofluorescence staining, the biggest difficulty is that it is difficult to coat the body of Caenorhabditis elegans in the outermost layer of skin Open, and then use the detergent Triton-X100 to permeate the cell membrane, effectively stain the subcellular structure inside and outside the cell, and stain the tissue and cells in the outer skin of Caenorhabditis elegans by destroying the outer skin of Caenorhabditis elegans , but none of these methods can maintain the complete domain morphology of C. elegans worms

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The materials used in this example: freeze-resistant glass slides; pre-made sheet-shaped dry ice with a size of 10-20 cm in length; pure acetone and methanol as the fixative, stored at -20°C, and the Slightly toxic coverage.

[0034] (1) Selection: C. elegans selects at least 2-4 discs of nematodes in each growth period without starvation in each phenotype. The staining of Caenorhabditis elegans and C. elegans with eggs in the Dauers period is not obvious.

[0035] (2) Quick freezing:

[0036] 1. Elute the selected C. elegans with nematode phosphate buffer M9buffer, and collect the C. elegans in a 1.5 mL test tube;

[0037] 2. Put the test tube on ice for 5 minutes, and suck up the supernatant with a pipette;

[0038] 3. Use 1mlddH 2 0 Repeat washing for Caenorhabditis elegans 3 times, and suck up the supernatant with a straw;

[0039] 4. Suspend Caenorhabditis elegans with 80ul volume fraction of 0.025% glutaraldehyde solution;

[0040] 5. Take out the freezing-resistant glass sl...

Embodiment 2

[0062] The materials used in this example: freeze-resistant glass slides; pre-made sheet-like dry ice with a size of 10-20 cm in length; 1xPBS containing 4% PAF as a fixing solution, operated in a ventilated place, stored at 20°C, and used When, cover the slight toxicity of the fixative.

[0063] (1) Selection: This step is basically the same as in Example 1;

[0064] (2) Quick freezing: this step is basically the same as in Example 1;

[0065] (3) Fixation: 1. Dispense 40ml of 1xPBS solution containing 4% PAF (paraformaldehyde) into 50ml centrifuge tubes, prepare one tube for each strain;

[0066] 2. Take out the quick-frozen sandwich-type glass slides from the dry ice, and quickly separate the two slides with both hands. At this time, the outer skin of C. elegans will be opened, keeping the body of C. elegans The integrity of the organization structure and form;

[0067] 3. Put the side of the glass slide without Caenorhabditis elegans quickly back to back and put it together; and i...

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Abstract

An immunofluorescent staining method for synapse sites in a nervous system is provided. The method includes steps of (1) selecting, (2) quick-freezing, (3) immobilizing and (4) antibody staining. The method is advantaged by a high freezing efficiency, a low cost, capability of effectively maintaining integrity of cell structures, good immobilizing effects, convenient staining operation and a high staining rate.

Description

Technical field [0001] The invention relates to an immunofluorescence staining method, in particular to an immunofluorescence staining method for synaptic sites in the nervous system. Background technique [0002] Caenorhabditis elegans is a very useful model animal with many advantages. Many scientific achievements have been obtained by using Caenorhabditis elegans as an animal model, such as the Nobel Prize for the programmed death mechanism of cell development in 2002 and 2006 The mechanism of the Nobel Prize's RNA interference phenomenon, the 2008 Nobel Prize-winning green fluorescent protein discovery and its application, etc. [0003] At present, many scientists have begun to use C. elegans for application development research, but when using C. elegans for immunofluorescence staining, the biggest difficulty is that it is difficult to coat the body of C. elegans in the outermost layer of skin Open, and then use detergent Triton-X100 to permeate the cell membrane, effectively...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30
CPCG01N1/30G01N2001/302G01N2001/305
Inventor 涂海军
Owner 武汉百齐生物技术有限公司
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