Goat anti rabbit igg cy3

Cells were grown in glass-bottom culture dishes at 37°C with 5% CO₂, according to the indicated timepoints. Then, cells were washed in phosphate buffered saline (PBS), fixed/permeabilized on ice for 30 minutes in fixation solution (2% paraformaldehyde in PBS), washed 4 times in PBS, and blocked for 15 minutes with 1% bovine serum albumin (BSA) in PBS. Next, the fixed cells were incubated at 4°C overnight with primary antibodies against PCNT (Abcam, Cambridge, MA), insulin (Abcam), and F-actin (Abcam), which were diluted at 1:400, 1:200, or 1:100 in PBS with 1% BSA. Then, cells were incubated for 1 hour in PBS containing 1% BSA and the appropriate secondary antibodies (bs-0295G, goat anti-rabbit IgG/Cy3, 1:100 dilution; bs-0358G, goat anti-guinea pig IgG/fluorescei isothiocyanate (FITC) 1:50 dilution; bs-0368G, goat anti-mouse IgM/Alexa flour 647, 1:100 dilution; Bioss, Beijing, China). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Abcam) and the slides were analyzed by microscopic examination (Olympus U-RFL-T, Tokyo, Japan). Confocal imaging was performed using a confocal microscope (Radiance 2000; BioRad, California) with a 60 × chromatic aberration free + infinity (CFI) plan Apo objective and a filter optimized for mCherry fluorescence.

The isolated CAFs or NFs were fixed in 4% paraformaldehyde. After permeabilization in 0.2% Triton X-100 and blocking in goat serum, the cells were probed with primary antibodies α-SMA (1:500, bsm-33187M, Bioss, Beijing, China) and vimentin (1:100, bsm-0756R, Bioss) overnight at 4°C. Subsequently, Goat Anti-Mouse IgG/FITC (1:100, bs-0296G-FITC, Bioss) and Goat Anti-rabbit IgG/Cy3 (1:100, bs-0295G-Cy3, Bioss) were applied. After nuclear counterstaining with DAPI, the cells were observed using a fluorescent microscope (Olympus, Tokyo, Japan).