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Method for acquiring antibody-labeled tissue single cells by laser microscopic cutting

A laser microdissection and antibody labeling technology, used in sampling, preparation of test samples, measurement devices, etc., can solve the problems of time-consuming acquisition of cells, difficult to effective cells, and many restrictions on cell size and tissue proportion. Achieve the effect of maintaining cellular RNA activity, good cellular RNA activity, and less RNA degradation

Inactive Publication Date: 2020-02-21
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing methods have problems such as time-consuming to obtain cells (more than three or four hours), more restrictions on cell size and tissue proportion in sorting, inability to accurately obtain a large number of target cells, and loss of cell location information in vivo.
Microdissection technology has the advantage of being able to locate and obtain cells, but it is rarely used in single cell research. Most of the existing research reports use fixed methods to process tissue cells. The single cells obtained by dissection are mainly used for PCR amplification and are difficult to obtain in large quantities. Efficient cells for single-cell sequencing

Method used

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  • Method for acquiring antibody-labeled tissue single cells by laser microscopic cutting
  • Method for acquiring antibody-labeled tissue single cells by laser microscopic cutting

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Obtaining adult rat spinal cord neurons

[0022] Such as figure 1 As shown, the specific steps for obtaining adult rat spinal cord neuron single cells are as follows:

[0023] (1) After adult rats are anesthetized, quickly take the spine and place it on dry ice for quick freezing; (from this step, the whole process of low temperature operation maintains the activity of tissue cells and prevents RNA degradation)

[0024] (2) intercept the lumbar enlargement part, slice it in a frozen microtome (thickness of 30 microns), and paste it on the special diaphragm for microdissection;

[0025] (3) Put the sliced ​​microdissection film in a drying box to dry for 10 minutes, and incubate with the Chat antibody that specifically labels motor neurons at 0-4°C for 10 minutes (the spinal cord motor neurons are labeled with specific markers). Antibody Chat, diluted at a high concentration of 1:100, the antibody carries green fluorescently labeled neuron cells), soaked and...

Embodiment 2

[0029] Example 2: Obtaining adult mouse substantia nigra dopaminergic neurons

[0030] (1) After the adult mouse is anesthetized, quickly remove the brain and place it on dry ice for quick freezing; (from this step on, the whole process of low temperature operation maintains the activity of tissue cells and prevents RNA degradation)

[0031] (2) Cut out the part of the substantia nigra, slice it in a frozen microtome (thickness 12 microns), and paste it on the special membrane for microdissection;

[0032] (3) Put the sliced ​​slices into a drying box to dry for 10 minutes, and incubate at 0-4°C for 10 minutes with a TH antibody that specifically labels motor neurons (the dopaminergic neurons in the substantia nigra are labeled with the specific antibody TH, at 1 : 100 high-concentration dilution, the antibody carries green fluorescent labeled neuron cells), soaked and washed with DEPC aqueous solution for 1 minute at 0-4°C; (immunohistochemical method antibodies quickly label...

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Abstract

The invention belongs to the technical field of biological tissue single cell acquisition, and discloses a method for acquiring antibody-labeled tissue single cells by laser microscopic cutting. The method comprises the following steps: treating animal tissues by a low-temperature method without fixing the animal tissues, quickly labeling and identifying target cells in situ in the animal tissuesby an optimized immunohistochemical method within a short time (0.5-1 hour), and cutting by combining a laser microscopic cutting method to obtain active target cells. The method can quickly and accurately acquire a large number of target cells in situ in the tissues, and can be used for special cells which are low in tissue proportion, large in cell volume, irregular in cell morphology and difficult to separate and culture; the position information of the obtained cells is clear, and the RNA activity of the cells can be well kept; and the method can be used for researches such as PCR and single cell sequencing with high requirements.

Description

technical field [0001] The invention relates to the technical field of obtaining single cells of biological tissues, in particular to a method for obtaining single cells of antibody-labeled tissues by laser microdissection. Background technique [0002] Single-cell sequencing can be better applied to the study of the mechanism of action of different cells in tissues. At present, the methods for obtaining single cells mainly include micropipulation, flow sorting, microdissection, and microfluidic sorting. Most of the existing methods have problems such as time-consuming to obtain cells (more than three or four hours), more restrictions on cell size and tissue proportion in sorting, inability to accurately obtain a large number of target cells, and loss of cell location information in vivo. . Microdissection technology has the advantage of being able to locate and obtain cells, but it is rarely used in single cell research. Most of the existing research reports use fixed met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/34
CPCG01N1/286G01N1/34G01N2001/2886
Inventor 何江虹徐慧栾成成顾晓松
Owner NANTONG UNIVERSITY