Rabbit anti choline acetyltransferase antibody

After differentiation, the cells were washed twice in PBS and fixed in PBS with 4% paraformaldehyde (Sigma-Aldrich, Saint Louis, MO, USA; 158127). Then, the cells were washed three times in PBS with 1% BSA (Sigma-Aldrich; A9418), permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA; T8787)/1% BSA/PBS with 5% normal goat serum (Jackson Immuno Research, Cambridge, UK; 005-000-121) for 45 min, and then incubated with rabbit anti-tyrosine hydroxylase antibody (dilution 1:600; AssayBioTech, Fremont, CA, USA; B0037) or with rabbit anti-choline acetyltransferase antibody (dilution 1:300; Proteintech, Manchester, UK; 20747-1-AP) in 1% BSA/PBS with 5% goat serum overnight at 4 °C. Subsequently, the cells were washed three times in PBS with 1% BSA and incubated with goat anti-rabbit IgG secondary antibody (DY405) (dilution 1:600; LSBio, Seattle, WA, USA; LS-C355899) in 1% BSA/PBS for 1 h in the dark at room temperature. Similar staining was done using a rabbit IgG isotype control (dilution 1:1500; Bioss, Woburn, MA, USA; bs-0295P) to determine any nonspecific binding. The cells were analyzed using flow cytometry (BD FACSAria II; BD FACSDiva Software V6.1.2).

Indirect labeling with a specific anti-TH or anti-CHAT antibody was used to identify the TH or CHAT protein-positive cells. After the differentiation, the cells were washed twice in PBS and fixed in PBS with 4% paraformaldehyde (Sigma-Aldrich; 158127). The cells were then washed three times in PBS with 1% BSA (Sigma-Aldrich; A9418), permeabilized with 0.3% Triton X-100 (Sigma-Aldrich; T8787)/1% BSA/PBS with 5% normal goat serum (Jackson Immuno Research, Cambridge, UK; 005-000-121) for 45 min and incubated with rabbit anti-tyrosine hydroxylase antibody (dilution 1:600; AssayBioTech, Fremont, CA, USA; B0037) or with rabbit anti-choline acetyltransferase antibody (dilution 1:300; Proteintech, Manchester, UK; 20747-1-AP) in 1% BSA/PBS with 5% goat serum overnight at 4 °C. The cells were washed three times in PBS with 1% BSA and incubated with goat anti-rabbit IgG secondary antibody (DY405) (dilution 1:600; LSBio, Seattle, WA, USA; LS-C355899) in 1% BSA/PBS for 1 h in the dark at room temperature. In order to determine any nonspecific binding, a similar staining was conducted using a rabbit IgG isotype control (dilution 1:1500; Bioss Antibodies; bs-0295P). Flow cytometry was used to analyze the cells (BD FACSAria II; BD FACSDiva Software V6.1.2).