Dsu spinning disk confocal scanner

Chick myogenic cells were rinsed with PBS and fixed in absolute methanol at −20 °C for 5 min. Then, they were permeabilized and blocked in PBS with 0.1% saponin, 1% BSA, and 5% horse serum for 30 min. Cells were incubated overnight at 4 °C with primary antibodies (all diluted 1:50 in the permeabilization and blocking solution). After incubation, cells were washed for 30 min in the permeabilization and blocking solutions and incubated for 1 h at 37 °C with Alexa Fluor-conjugated secondary antibodies (all diluted 1:100 in the permeabilization and blocking solution). Nuclei were labeled with DAPI, Hoechst, or NucSpot. Cells were mounted in Prolong Gold (Molecular Probes) and examined with either an Axiovert 100 microscope (Carl Zeiss, Jena, Germany) coupled to an Olympus DP71 high-resolution camera, a Leica TCS SPE laser scanning confocal microscope (Leica, Wetzlar, Germany), or a DSU Spinning Disk confocal scanner mounted on an inverted fluorescent microscope (Olympus, Shinjuku, Japan). Control experiments with no primary antibodies showed only a faint background staining (data not shown). Image processing was performed using Fiji software, version 1.53q [9 (link)] and figure plates were mounted with Adobe Photoshop software, version 7.0.1 (Adobe Systems Inc., Mountain View, CA, USA).