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Stationary liquid and fixed cell method and application

A fixative fixation and fixative technology, applied in the field of immunofluorescence, can solve the problem that the fluorescence effect needs to be improved, and achieve the effect of significant fluorescence effect, prevention of antigen loss, and beautiful and accurate fluorescence images.

Inactive Publication Date: 2020-01-14
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fluorescence effect of immunofluorescence detection of cells fixed with 4% paraformaldehyde needs to be improved

Method used

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  • Stationary liquid and fixed cell method and application
  • Stationary liquid and fixed cell method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] This example is the induction of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes

[0049]1. HiPSC-U1 culture method: freeze-thaw Matrigel from -80°C to 4°C overnight, aliquot Matrigel the next day and add 100 μl Matrigel to 10ml DMEM / F-12 for culture in a 15ml centrifuge tube, store Store in the refrigerator at 4°C for later use. Every day, replace each well with a new medium: 4ml TeSR-E8 medium until the number of hiPSCs reaches about 75%.

[0050] 2. On the 0th day of differentiation, discard the old medium, replace and add 4ml (12μM CHIR99021+RPMI / B-27minus insulin) medium to each well, and return the multi-well plate to 37°C, 5% CO 2 Incubate in the incubator for 24h.

[0051] 3. On the first day of differentiation, discard the old medium, add 4ml of RPMI / B-27 medium (without insulin) to each well, and return the multi-well plate to 37°C, 5% CO 2 Incubate in the incubator for 24h.

[0052] 4. On the third day of differentiation, 72 hours after ...

Embodiment 2

[0056] This embodiment is the immunofluorescent staining of cardiomyocytes in Example 1

[0057] (1) Digestion:

[0058] Aspirate off the old medium and wash the differentiated cells twice with 2 ml of PBS per well. Aspirate off the PBS, add 2ml (0.25% (wt / vol) trypsin-EDTA) dissociative enzyme to dissociate the cells, and digest at 37°C, 5% incubator for 5min.

[0059] (2) Beating:

[0060] Cells were singulated by pipetting 5-10 times with a 1 ml pipette. Then transfer the pipetted single cell suspension to a 15ml centrifuge tube containing 4ml RPMI 20.

[0061] (3) Counting:

[0062] Take 10 μl of the cell suspension and count the cells with a hemocytometer; and centrifuge the cell suspension at a speed of 1000 r / min for 4 min at room temperature. After centrifugation, the supernatant was discarded using a 1 ml pipette.

[0063] (4) Incubating cells:

[0064] 2ml / well cell suspension (1×10 4 cells / ml) in 6-well culture dishes containing (Matrigel coverslips). at 37...

Embodiment 3

[0084] The immunofluorescent stained samples of Example 2 and Comparative Example 1 were placed under an inverted fluorescence microscope to observe and collect images.

[0085] figure 1 The cardiomyocyte immunofluorescence 40X optical microscope picture provided for Comparative Example 1 of the present invention;

[0086] figure 2 The 40X optical microscope image of cardiomyocyte immunofluorescence provided for Comparative Example 1 of the present invention. Depend on figure 1 It can be seen that: figure 1 Fluorescence can accurately locate the cell nucleus (DAPI), but the cell structure is not clear, and the sarcomere structure of the corresponding specific protein (cTnT, αAtinin) in cardiomyocytes cannot be accurately seen, and the background of the picture is blurred under the condition of good focus unclear, affecting the overall effect; figure 2 Under the action of the new fixative, the focus can be aligned, the nucleus (DAPI) can be accurately positioned, and the...

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Abstract

The invention relates to the immunofluorescence technology field, and especially relates to a stationary liquid and fixed cell method and application. The invention discloses a stationary liquid. A liquid A is cold methanol with a volume fraction of 90%, and a liquid B is paraformaldehyde with a volume fraction of 1%. The stationary liquid is beneficial to cell climbing. Cells are not easy to falloff, the number of the cells is large during immunohistochemistry, a dosage of paraformaldehyde in the stationary liquid is small, antigen losses can be prevented, normal structures of the cells canbe maintained, and antigens can be kept in a state of combining with antibodies as much as possible so that a fluorescence effect is remarkable during immunofluorescence detection, and a fluorescenceimage is attractive and accurate. Besides, compared with other stationary liquids, the stationary liquid is economical and easy to obtain, does not have flammable and explosive hazards, can be operated at a room temperature, and does not require a special experimental environment.

Description

technical field [0001] The invention relates to the technical field of immunofluorescence, in particular to a fixative solution and a method and application of fixing cells. Background technique [0002] The pretreatment for immunofluorescence analysis is to fix the tissue cells. From the perspective of immunohistochemical technology, the role of fixation is not only to coagulate intracellular proteins, but also to minimize or terminate the reaction of exogenous enzymes and endogenous enzymes; to prevent autolysis of cells, so as not to cause antigens to diffuse into the interstitial tissue; Maintain the inherent shape and structure of the tissue; more importantly, maintain the antigenicity of the tissue or cells, not only to prevent the inactivation of the antigen, but also to prevent the diffusion of the antigen, so that the immunohistochemical staining does not produce too deep Therefore, the choice of fixative is very important, which determines whether accurate and usa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/533G01N33/541
CPCG01N21/6428G01N21/6458G01N33/533G01N33/541G01N2021/6439
Inventor 苗小敏汤亚东张焜陈家盈周颖梁大锡李卓刚罗竣仁
Owner GUANGDONG UNIV OF TECH
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