Sp8x wll upright confocal microscope

For immunostaining, cells were seeded on glass coverslips coated with 0.15% gelatin. In indicated experiments, cells were treated with 0.2 U/ml thrombin or 10 μM Y-27632 for 10 min. Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 30 min and incubated with blocking solution (1% bovine serum albumin, 0.1% Triton X-100 in PBS) for 1 h at room temperature. Cells were exposed to primary antibodies overnight at 4°C. After washing three times with 0.1% Triton X-100 in PBS for 15 min, secondary antibodies were applied for 1 h at RT. After washing three times with PBS for 15 min, cells were embedded in Fluoromount (Southern Biotech) and analyzed with a Leica SP8X WLL upright confocal microscope (Leica).

Microsurgical preparation of the cremaster muscle and in vivo microscopy was performed as described previously (Rehberg et al., 2010 (link)). Briefly, mice were anesthetized using a ketamine/xylazine mixture (100 mg/kg ketamine and 10 mg/kg xylazine), administrated by intraperitoneal injection. The right cremaster muscle was exposed through a ventral incision of the scrotum. The muscle was opened ventrally in a relatively avascular zone, using careful electrocautery to stop any bleeding, and spread over the pedestal of a custom-made microscopy stage. Epididymis and testicle were detached from the cremaster muscle and placed into the abdominal cavity. Throughout the procedure as well as after surgical preparation during in vivo microscopy, the muscle was superfused with warm buffered saline.
After in vivo microscopy, the tissue was fixed in 2% paraformaldehyde and immunostained as whole mount. After washing three times with PBS for 15 min, cells were embedded in Fluoromount (Southern Biotech) and analyzed with a Leica SP8X WLL upright confocal microscope (Leica). Images were analyzed offline using LAS AF software (Leica, Germany), ImageJ (NIH), and IMARIS (Oxford Instruments).