VSMCs were grown on coverslips at a density of 2 × 104 per well with appropriate treatment and fixed in 4% formaldehyde for 30 min. After washing with PBS for three times, cells were permeated with 1 ml Triton X-100 at room temperature for 20 min. Thereafter, cells were incubated with primary antibodies against MCPIP1 (1:200 diluted, ab97910, Abcam, Cambridge, MA, U.S.A.), α-SMA (1:200 diluted, ab32575), rabbit IgG-Isotype Control (Alexa Fluor® 647) (1:500 diluted, ab199093) and rabbit IgG Isotype control (FITC) (1:100 diluted, ab223339) at 4°C overnight and subsequently incubated with FITC/Alexa Fluor® 647-labeled goat anti-rabbit IgG (H+L) secondary antibody (1:1000 diluted, ab6717/ab150115, Abcam, Cambridge, MA, U.S.A.) at a dilution of 1:200 for 2 h at 37°C. Unbound antibodies in each step were removed with PBS for three times. After staining with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and a final rinse with PBS, the coverslips were mounted inversely on to slides with 95% glycerol and observed under a fluorescence microscope (Olympus Corporation, Japan) and photographed.
Ab199093
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab199093 is a lab equipment product offered by Abcam. It is a functional tool designed for laboratory use, but a detailed description is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
2 protocols using ab199093
1
Immunofluorescence Staining of VSMCs
2
Flow Cytometric Analysis of PD-L1 and PD-1 Expression
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The cells were digested by pancreatin (C125C1, NCM Biotech, Suzhou, Jiangsu, China,) and incubated with PD‐L1‐Alexa Fluor 647 (1:100; ab209960; Abcam, Cambridge, UK,) and the control were incubated with isotype (1:100; ab199093; Abcam) at 4°C for 30 min. After incubation, the cells were washed twice and suspended in phosphate‐buffered saline (PBS). The cells were analyzed by Flow Cytometry (BD Bioscience).
The mouse lung tissues were digested with collagenase I (A004194, Sangon Biotech, Shanghai, China). Cells were stained with fluorescently labeled antibodies. Then cells were analyzed by Flow Cytometry (BD Bioscience), and the data were analyzed using the FlowJo10 software (BD Bioscienc). The following antibodies were used: anti‐CD45 PE/Cy7‐conjugated (1;100;103113;BioLegend, San Diego, CA, USA), anti‐CD3ε PerCP/Cy5.5‐conjugated (1:100;100327; BioLegend), anti‐PD‐1‐APC‐conjugated (1:100; 135209; BioLegend).
The mouse lung tissues were digested with collagenase I (A004194, Sangon Biotech, Shanghai, China). Cells were stained with fluorescently labeled antibodies. Then cells were analyzed by Flow Cytometry (BD Bioscience), and the data were analyzed using the FlowJo10 software (BD Bioscienc). The following antibodies were used: anti‐CD45 PE/Cy7‐conjugated (1;100;103113;BioLegend, San Diego, CA, USA), anti‐CD3ε PerCP/Cy5.5‐conjugated (1:100;100327; BioLegend), anti‐PD‐1‐APC‐conjugated (1:100; 135209; BioLegend).
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