Prolon gold antifade reagent

Cells were plated at 2×10⁵ cells per chamber in Lab-Tek II CC2 chamber slide (Nunc) for 24 hours. Cells were fixed 10 minutes at room temperature with 4% PFA, blocked 1 hour with 1% BSA and stained for extracellular secreted fibronectin (1:200) 1 hour at room temperature. After extensive wash, anti-Alexa546 was incubated an additional 1 hour at room temperature. Slides were mounted using Prolon Gold antifade reagent (Invitrogen) and observed using a fluorescent microscope (AXIO, Zeiss) with a 4X magnification.

hTERT-BJ fibroblasts were fixed in 3% paraformaldehyde for 20 min at room temperature. Thereafter, cells were permeabilized with 0.25% Triton X-100 for 10 min, washed in PBS, blocked by 3% BSA and 5% goat serum in PBS for 1 h, incubated with primary antibodies for 90 min at room temperature, washed four times in PBS, and incubated with secondary antibodies for 1 h. The following primary and secondary antibodies were used at the indicated dilutions and concentration: anti-p53 (1:200; mouse IgG DO-1), anti-CD44 (4 μg/mL; Hermes1), iASPP antiserum (1:200), Alexa Fluor 488 goat anti-mouse (1:1000), and Alexa Fluor 594 goat anti-rabbit (1:1000). Slides were mounted with ProLon gold antifade reagent (Invitrogen), and photographs were taken with a Zeiss Axioplan 2 immunofluorescence microscope and/or a Zeiss LSM 700 confocal module.