Aug 10, 2023
ChAT Immunofluorescent Staining
- 1University of Ottawa
Protocol Citation: Haley Geertsma 2023. ChAT Immunofluorescent Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxbz4gx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 10, 2023
Last Modified: August 10, 2023
Protocol Integer ID: 86350
Keywords: choline acetyltransferase, ChAT
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.
ChAT Immunofluorescent Staining
ChAT Immunofluorescent Staining
2d 4h 35m
2d 4h 35m
Wash tissue slices in 1X phosphate buffered saline (PBS) for 5 minutes. Repeat x2.
5m
Incubate in blocking buffer (10% horse serum, 0.5% gelatin, 0.1% triton X-100 in 1X PBS) for 2 hours at room temperature.
2h
Incubate in primary antibody (goat anti-ChAT (RRID:AB_2079751) at 1:200 dilution in blocking buffer) at 4oC for 48 hours.
2d
Wash tissue with 1X PBS for 5 minutes. Repeat x4.
5m
Incubate in secondary antibody for 2 hours at room temperature.
2h
Wash tissue with 1X PBS for 5 minutes. Repeat x4.
5m
Mount free-floating sections on SuperFrost+ slides (if staining free-floating tissue) and let dry at room temperature for 15 minutes.
15m
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.
5m