Axioplan 2 immunofluorescence microscope

Approximately 95% confluent infected NMuMG monolayers were analyzed by immunofluorescence 36-48 h post-infection or 48 h post-siRNA transfection as described [34 (link)]. Cells were counterstained with 4’,6’-diamidino-2-phenylindole (DAPI) to visualize the nuclei. A Zeiss Axioplan 2 immunofluorescence microscope was used with the Zeiss 20×, 40× or 63× objective lens and photographing at ambient temperature in the presence of immersion oil. Images were acquired with a Hamamatsu C4742-95 CCD digital camera and the acquisition software QED Camera Plugin v1.1.6 (QED Imaging Inc.) and Volocity^® (PerkinElmer). Images taken were processed with Adobe Photoshop 6.0 or CS2 to reduce file size.

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.