Sp5 aobs upright 2 laser scanning confocal microscope

C2C12 cells were treated as indicated, fixed, permeabilized, blocked, and incubated sequentially with primary antibodies (anti-Smad2/3 or anti-IRF3) and Alexa-labeled secondary antibodies with extensive washing. Slides were then mounted with Vectorshield and stained with DAPI. Images were obtained and analyzed using a Leica SP5 AOBS Upright 2 laser scanning confocal microscope. For F-actin staining, HaCaT cells were fixed, washed, and then incubated with Alexa-546 phalloidin at a 1:500 dilution. Images were obtained and analyzed by a Leica DMI 4000 B inverted microscope.

Cells were fixed in 3.7% paraformaldehyde for 5 min, permeabilized in PBS with 0.2% Triton-X100 (PBS-T) for 5 min, and blocked with 5% BSA in PBS for 2 h at room temperature. Cells were incubated with rabbit-anti GFP (1:100, Invitrogen) and mouse anti-Flag (M2) at 1:100 diluted in 5% BSA/PBS overnight at 4°C, washed with PBS at room temperature, and then incubated with secondary Alexa Fluor-conjugated secondary (Invitrogen) in 5% BSA/PBS for 2 h at room temperature in the dark. Slides were mounted with Vectorshield and stained with DAPI. Immunofluorescence images were obtained using a Leica SP5 AOBS Upright 2 laser scanning confocal microscope. Changes in cell morphology were observed by phase contrast microscopy using a Leica microscope DMI5000, and analyzed using Photoshop CS5.