Confocal Microscopy Sample Clauses

Confocal Microscopy. NIH-3T3 cells transiently transfected with GFP-tagged constructs were grown on collagen I-coated culture slides (BD Biosciences), and 48 h after transfection cells were fixed with 4% PFA at room temperature (RT) for 10 minutes. Following one wash with PBS+Ca2+, cells were blocked in blocking buffer (PBS+Ca2+ + 1% goat serum (Invitrogen) and 0.1% Triton-X-100) for 30 minutes at RT. Cilia were labeled with rabbit anti-Arl13b antibody (1:500; ProteinTech) overnight in blocking buffer. Following primary antibody incubation, cells were washed three times in blocking buffer and incubated with Alexa 546 fluorophore (red) conjugated anti-rabbit secondary antibody (Invitrogen) in blocking buffer for 1h at RT. After a single rinse, cell nuclei were stained with DAPI (USB Affymetrix) for 10 min. Following two additional rinses, cells were mounted onto slides using Vectashield mounting medium (Vector Laboratories) and sealed. Images were captured using an Olympus FV1000 confocal microscope (Olympus).