Goat anti rabbit igg h l alexa fluor 647 secondary antibody

Samples were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton. After being blocked with 5% bovine serum albumin, the samples were incubated with the following reagents: YF®488-phalloidin (US EVERBRIGHT, China, 1:200), anti-α-SMA (Abcam, UK, 1:500), and anti-MRTF-A (Thermo Fisher, USA, 1:200). For the primary antibodies without conjugated fluorescent labels, samples were further incubated with goat anti-rabbit IgG H&L Alexa Fluor® 647 secondary antibody (Abcam, 1:1,000). Cell nuclei were stained with DAPI (Beyotime, China). The staining was observed with a confocal laser scanning microscope (LSM880 WITH AIRYSCAN).

1 × 10⁵ cells/per 24 well plate were seeded on glass coverslips incubated overnight to form a confluent monolayer. Cisplatin was added at IC⁶⁰ concentrations determined by previous experiments for 48 h. Cells were fixed, permeabilised, blocked and stained with the Cleaved Caspase-3 antibody ASP¹⁷⁵ (Abcam #ab2303) (1:2000) followed by the goat anti-rabbit IgG H&L Alexa Fluor® 647 secondary antibody (Abcam, Cambridge, UK). Coverslips were removed from the 24-well plate and were mounted onto microscope slides using FluorSave mounting medium (Calbiochem #345789). Slides were imaged using the Cytation™ 3 Multimode Imager. Subsequent subpopulation analyses were performed as described.