Ab683 clone 1g12

Anal tubes were isolated and fixed in ice-cold acetone for 10 min, then washed with PBS overnight at 4 °C and rewashed for 4 h with a change of PBS per hour. Cryosections with a 10-μm thickness were used. The non-specific binding of primary antibodies was blocked by incubation with PBS containing 1% BSA and 5% non-immune goat serum for 1 h. Incubation was carried out overnight at 4 °C with a rabbit polyclonal antibody to TMEM16A (ab53212, abcam) diluted 1:200, together with a rat anti-c-Kit antibody (ACK2, Chemicon, 1:100) or mouse monoclonal Myh11 antibody (ab683 clone 1G12, 1:200; abcam). After washing in PBST, sections were incubated with a Alexa Fluor 555-conjugated goat anti-Rabbit antibody (Sigma) diluted 1:250 and a FITC-conjugated goat anti-rat antibody (Invitrogen) diluted 1:250 or a Alexa Fluor 488–conjugated goat anti-mouse antibody (Cell signaling technology) for 1 h. 4,6-diamidino-2-phenylindole was used for nuclear staining. Immunoreactivity was evaluated using a FV1000 confocal laser scanning microscope system (Olympus).

Protein distribution was assessed by immunohistochemistry as described (Lu et al., 2021a (link)). Uterine cryosections were 8 μm, and antibodies were mouse monoclonal antibody to MYH11 (ab683 clone 1G12, 1:200; Abcam) and Alexa Fluor 488-conjugated goat anti-mouse IgG (Cell Signaling Technology, dilution 1:500). Immunoreactivity was evaluated using a Leica TCS SP8 confocal laser scanning microscope system (Leica Microsystems Inc., Buffalo Grove, IL, United States).