For IF, the GLI1 antibody was used at 10 ug/ml (Hu, 2010).
Protocol: Cells were washed in PBS and fixed in 4% PFA, pH 7.4 (20g PFA in 500 ml PBS) for 10 min. The fixed cells were permeabilized in 0.5% Triton X-100 (in PBS) for 5 min and blocking in 50 ul of 20% HINGS (heat inactivated normal goat serum diluted in PBS) for 1 hr. The GLI1 antibody was incubated at 37 degrees C for 1 hr or at 4 degrees C overnight. Following 3 X 5 min washes in PBS, an AlexaFluor488 or 594-conjugated goat anti-rabbit antibody was used as a second step (diluted in 20% HINGS at 1:1000 for 1 hr at room temperature i the dark). Following 3 X 5 min washes in PBS, the slides were mounted with DABCO (1,4-diazabicyclo[2.2.2]octane) anti-fading agent for fluorescence microscopy.
