Axiocam mr color camera

Immunofluorescence. At 24 h p.i., PC3 cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) for 5 min and blocked with 1% bovine serum albumin in PBS (PBSA) for 10 min with PBS washes between each step. Cells were then incubated for 45 min at room temperature with primary antibodies diluted in PBSA and washed two times with PBS, followed by incubation with secondary antibody diluted in PBSA for 45 min and two additional PBS washes.
Coverslips with labeled cells were mounted with ProLong Gold antifade reagent with DAPI (4,6-diamidino-2-phenylindole dihydrochloride; Invitrogen Life Technologies) on slides. Each coverslip was then examined on a Zeiss Axiovert 200 inverted microscope equipped with fluorescence optics to determine the number of cells expressing nuclear HIF-1a. Representative pictures were taken by a Zeiss AxioCam MR color camera using AxioVision software (4.8.2). Images were prepared using Adobe Photoshop and Illustrator software (Adobe Systems).