ChAT Immunofluorescent Staining
Haley Geertsma
Abstract
This protocol is designed for 3-day choline acetyltransferase (ChAT) staining using the EMD Millipore AB144P (RRID:AB_2079751) antibody. Tissue stained with this protocol include 40μm free-floating mouse brain and spinal cord sections, and 20μm pre-mounted retina sections. All tissue was from mice perfused with 10% buffered formalin.$$
Steps
ChAT Immunofluorescent Staining
Wash tissue slices in 1X phosphate buffered saline (PBS) for 5 minutes. Repeat x2.
Incubate in blocking buffer (10% horse serum, 0.5% gelatin, 0.1% triton X-100 in 1X PBS) for 2 hours at room temperature.
Incubate in primary antibody (goat anti-ChAT (RRID:AB_2079751) at 1:200 dilution in blocking buffer) at 4oC for 48 hours.
Wash tissue with 1X PBS for 5 minutes. Repeat x4.
Incubate in secondary antibody for 2 hours at room temperature.
Wash tissue with 1X PBS for 5 minutes. Repeat x4.
Mount free-floating sections on SuperFrost+ slides (if staining free-floating tissue) and let dry at room temperature for 15 minutes.
Coverslip with fluorescent mounting medium and #1.5 coverslips. Outline coverslip with clear nail polish and store at 4oC or -20oC depending on length of storage.
