Alexa fluor 555 rabbit anti mouse secondary antibody

Myocytes were plated onto poly-L-lysine-coated coverslips, fixed with 3.7% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C. Slides were then washed and incubated with Alexa Fluor 555 rabbit anti-mouse secondary antibody (Molecular Probes). Secondary antibodies were washed and coverslips mounted onto slides. Images were acquired using a Zeiss 710 (Carl Zeiss) laser-scanning confocal microscope and 40x and 63x oil immersion objectives.

Arteries were cut longitudinally and fixed with 4% paraformaldehyde in PBS for 1 hr. Following a wash in PBS, arteries were permeabilized with 0.2% Triton X-100, blocked with 5% goat serum and incubated overnight with PKD2 primary antibody (Rabbit mAB 3374 CT-14/4: Baltimore PKD Center) at 4°C. Arteries were then incubated with Alexa Fluor 555 rabbit anti-mouse secondary antibody (1:400; Molecular Probes) and 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (1:1000; Thermo Scientific) for 1 hr at room temperature. Arteries were washed with PBS and mounted in 80% glycerol solution. DAPI and Alexa 555 were excited at 350 nm and 555 nm with emission collected at ≤ 437 nm and ≥ 555 nm, respectively, using a Zeiss LSM 710 laser-scanning confocal microscope.

Arteries were cut longitudinally and fixed with 4% paraformaldehyde in PBS for 1 hr. Following a wash in PBS, arteries were permeabilized with 0.2% Triton X-100, blocked with 5% goat serum and incubated overnight with PKD2 primary antibody (Rabbit mAB 3374 CT-14/4: Baltimore PKD Center) at 4°C. Arteries were then incubated with Alexa Fluor 555 rabbit anti-mouse secondary antibody (1:400; Molecular Probes) and 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (1:1000; Thermo Scientific) for 1 hr at room temperature. Segments were washed with PBS, oriented on slides with the endothelial layer downwards and mounted in 80% glycerol solution. DAPI and Alexa 555 were excited at 350 nm and 555 nm with emission collected at ≤437 nm and ≥555 nm, respectively.