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An Immunofluorescent Staining Method for Synaptic Sites in the 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, etc., to improve the fixation effect and fixation rate, The effect of maintaining integrity and facilitating dyeing

Active Publication Date: 2018-03-02
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] Materials used in this example: freeze-resistant glass slides; dry ice pre-made into sheets with a size of 10-20 cm in length; pure acetone and methanol are used as fixatives, stored at -20°C, and when used, the Slightly toxic overlay.

[0034] (1) Selection: Caenorhabditis elegans select at least 2-4 plates of nematodes in each growth stage without starvation in each phenotype, and the staining of C. elegans in the Dauers stage and egg-bearing C. elegans is not obvious.

[0035] (2) quick freezing:

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

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

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

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

[0040] 5. Ta...

Embodiment 2

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

[0063] (1) selection: this step is basically the same as Example 1;

[0064] (2) quick freezing: this step is basically the same as embodiment 1;

[0065] (3) Fixing: 1. 40ml of 1xPBS solution containing mass fraction of 4%PAF (paraformaldehyde) is subpackaged in 50mL centrifuge tubes, and one tube is prepared for each strain of worms;

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

[0067] 3. Put the sides of the slides without Caenor...

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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 of a nervous system synaptic site. 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 programmed death mechanism of cell development won the Nobel Prize in 2002, and won the 2006 The mechanism of the RNA interference phenomenon of the Nobel Prize, the discovery and application of the green fluorescent protein that won the Nobel Prize in 2008, etc. [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 ...

Claims

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Application Information

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Patent Type & Authority Patents(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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