Tcs2 confocal microscope

Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, blocked with PBS containing 2% bovine serum albumin (BSA) plus 5% normal goat serum for 1 h, and incubated overnight at 4°C with mouse anti-desmin antibody (1:50; DakoCytomation, Carpinteria, CA), rabbit anti-myogenin (1:50; Santa Cruz Biotechnology), or mouse anti–myosin heavy chain (1:20; Developmental Studies Hybridoma Bank). After three washes with PBS, cells were incubated with Alexa Fluor 546–conjugated or Alexa Fluor 488–conjugated secondary antibodies (Molecular Probes, Eugene, OR) for 1 h at room temperature and washed with PBS. Nuclei were counterstained with either 4′,6-diamidino-2-phenylindole (DAPI; 100 ng/ml) or TOTO-3 (2.4 nM; Molecular Probes). Slides were then mounted in Vectashield (Vector Laboratories, Burlingame, CA). Cells were visualized using an Eclipse E-1000 microscope (Nikon, Melville, NY) and photographed using a Hamamatsu digital camera (Hamamatsu, Bridgewater, NJ), and images were acquired using Openlab software, version 3.1.5 (Improvision, PerkinElmer, Waltham, MA). For confocal imaging, all images were acquired with a 40×/1.25 numerical aperture oil immersion objective using a TCS2 confocal microscope (Leica, Wetzlar, Germany).