Alexa fluor 555 antibodies

PC-3U cells were cultured in six-well dishes in the presence of RPMI-1640 medium supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin and streptomycin. Cells were transfected with the respective plasmids; after 24 h, cells were starved in RPMI-1640 medium supplemented with 1% FBS, 1% L-glutamine, and 1% penicillin and streptomycin, and after an additional 18 h, cells were stimulated with 10 ng/ml of TGFβ1.
Cells were washed in PBS and fixed in 4% paraformaldehyde, followed by permeabilization in 2% Triton X-100. Cells were blocked in 5% BSA. After blocking, cells were incubated with primary antibody for 1 h, followed by incubation with secondary donkey anti-rabbit Alexa Fluor 555 antibodies or goat anti-mouse Alexa Fluor 488 antibodies (Invitrogen). After staining, cells were mounted in medium with 4’,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei. Images were taken with a confocal microscope with an oil immersion 63X lens. Zen software was used to analyze the data.

Approximately 2.4 × 10⁴ MCF-7 cells or 1.2 × 10⁴ T47D cells were seeded per well in a 48-well plate. The following day, cells were treated with reduced growth media containing 4-OHT, pyrvinium or DMSO. After 48 h of treatment, cells were fixed in 4% (w/v) paraformaldehyde (PFA). Cells were washed three times in PBS, then permeabilized for 15 min in PBS containing 0.1% (v/v) Triton X-100. Cells were washed three times in PBS, then were blocked for 1 h in PBS containing 3% (w/v) BSA. Cells were incubated overnight at 4 °C with Ki67 antibodies (ThermoFisher Scientific, cat # RM-9106-S1, 1:200) diluted in PBS containing 3% (w/v) BSA. Cells were washed three times in PBS, then incubated for 1 h with donkey anti-rabbit Alexa Fluor^® 555 antibodies (Invitrogen, cat # A-31572, 1:500) and DAPI (Sigma, cat # D9542, 1:1000) diluted in PBS containing 3% (w/v) BSA. Cells were washed three times in PBS, then imaged using an Invitrogen EVOS^® M5000 Imaging System with a 10× objective. The percentage of Ki67-positive cells was determined using the “cell counter” plugin in ImageJ version 2.0.0 software.