Immunofluorescence assays were performed as follows: cells (1 × 105 cells/well) were seeded into 12-well plates with a round coverslip (Biosharp) and cultured for 12–24 h. The cells were then fixed with 4% paraformaldehyde (YuanYe Bio-Technology) for 20 min at room temperature and then permeabilized with 0.5% Triton X-100 (Solarbio) for 15 min at room temperature. Nonspecific antibody binding was blocked by washing the cells with 5% normal goat working serum (Solarbio) for 1 h at room temperature, and the cells were incubated with NKX2.5 antibody (1:200, Abcam), GATA4 antibody (1:100, Proteintech), β-catenin (1:500, Proteintech), and cTnT antibody (1:500, Abcam) overnight at 4 ℃. The secondary antibody (Alexa Fluor 568, Invitrogen) was incubated with the cells at room temperature for 1.5–2 h, and DAPI (1:5000, Beyotime Biotechnology) was added for 10 min to stain the nucleus. Images were acquired using a fluorescence microscope (Carl Zeiss).
Round coverslip
Manufactured by Biosharp
Sourced in China
The round coverslip is a flat, circular piece of glass or other transparent material used in various laboratory applications. Its core function is to cover and protect samples on microscope slides, enabling clear visualization and observation.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Solarbio (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Yuanye Bio-Technology (1 mentions)
Nkx2.5 antibody, by Abcam (1 mentions)
β catenin, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Dmi3000b fluorescence microscope, by Leica (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by ABclonal (1 mentions)
4 6 diamidino 2 phenylindole dapi solution, by Beyotime (1 mentions)
2 protocols using round coverslip
1
Immunofluorescence Assay for Cell Characterization
2
Immunofluorescence Staining of Cells
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Cells were plated on the round coverslip (Biosharp, Hefei, Anhui, China) at a density of 105 cells/coverslip. After washing with PBS, cells were fixed with 4% formaldehyde for 10 min, permeabilized with 0.5% Triton X‐100 for another 10 min and blocked with 5% bovine serum albumin. The coverslips were then incubated with primary antibodies at 4°C overnight and Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (#AS039, dilution 1:250, ABclonal, Wuhan, Hubei, China) at room temperature for 2 h. Nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) solution (#C1002, Beyotime) at room temperature for 15 min, and the images were captured by a DMI3000B fluorescence microscope (Leica, Wetzlar, Hessen, Germany).
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