Alexa fluor 488 conjugated donkey anti rabbit igg h l

Anal tissues were isolated, and the skeletal muscle fibers were removed, immediately followed by embedded in Optimal cutting temperature compound (Bio-Tek). Cryosections with a 10-μm thickness were fixed in pre-cooled acetone for 10 min and washed with PBS three times at room temperature. The nonspecific binding of primary antibodies was blocked by incubation with PBST (0.3% Triton in PBS) containing 1% BSA for 1 h. Incubation was carried out overnight at 4°C with a rabbit polyclonal antibody to TMEM16A (ab53212, 1:100; Abcam) and a goat polyclonal antibody against c-Kit (AF332, 1:20; R&D systems). The specificity of these antibodies has been established by vendors and others. After washing in PBS, cells were incubated with an Alexa Fluor 488-conjugated donkey anti-Rabbit IgG H&L (ab150061, 1:500; Abcam) or an Alexa Fluor 594-conjugated rabbit anti-goat IgG (H+L) (A27016, 1:500; Life tech) for 1 h. Negative controls were performed by omitting the primary antibodies. Immunoreactivity was evaluated using a Leica TCS SP5 confocal laser scanning microscope system (Leica Microsystems Inc., Buffalo Grove, IL).