Fluoroshield mounting medium with dapi

Tissue or cell chamber slides were washed with PBS, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin. The slides were then incubated with primary antibodies, followed by incubation with Alexa Fluor-conjugated secondary antibodies. Fluoroshield mounting medium with DAPI (VECTOR Laboratories, Burlingame, CA) was used to cover the slides. Images were visualized under an Olympus inverted microscope equipped with a charge couple camera.

Apoptotic cell death was analyzed using the in situ cell death detection kit (Roche). Cells were immobilized onto slides, fixed in 4% paraformaldehyde for 1 h at room temperature, permeabilized with 0.1% Tween 20 (in phosphate-buffered saline [PBS]), and then incubated with the TUNEL reagents (TdT enzyme-dUTP, 1:10) for 1 h at 37°C. The slides were mounted in Fluoroshield mounting medium with DAPI (Vector Laboratories) and analyzed under an Olympus BX61 DSU confocal microscope with a 60× objective. Dual fluorescence properties were observed using a multiwavelength filter as described above. All treatments were replicated three times.