Sep 09, 2024
ChAT Immunofluorescent Staining of medulla oblongata fixed sections
Forked from ChAT Immunofluorescent Staining
- 1German Center for Neurodegenerative Diseases (DZNE)
Protocol Citation: Pietro La Vitola 2024. ChAT Immunofluorescent Staining of medulla oblongata fixed sections. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32nzpv25/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: September 06, 2024
Last Modified: September 09, 2024
Protocol Integer ID: 107044
Keywords: choline acetyltransferase, ChAT, immunofluorescence
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol is designed for choline acetyltransferase (ChAT) staining using the Millipore AB144P (RRID:AB_2079751) antibody. Tissue stained with this protocol include 30μm free-floating mouse brain sections. All tissue was from mice perfused with 4% PFA.
ChAT Immunofluorescent Staining of medulla oblongata fixed sections
ChAT Immunofluorescent Staining of medulla oblongata fixed sections
2d 4h 35m
2d 4h 35m
Wash tissue slices in tris-buffer saline (TBS: 0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes. Repeat x3.
30m
Incubate in in 10% Methanol + 3% H2O2 in TBS for 20 min at room temperature
20m
Wash tissue slices in tris-buffer saline (TBS: 0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes. Repeat x3.
30m
Incubate in blocking buffer (5% donkey serum, 2% BSA, 0.5% triton X-100 in TBS) for 1 hour at room temperature.
1h
Incubate with anti-ChAT primary antibody (RRID:AB_2079751) at 1:500 dilution in 50% blocking solution for 48 hours at 4°C.
2d
Wash tissue slices in TBS-T (TBS+ 0.25% Triton x-100) for 10 minutes. Repeat x3.
30m
Incubate in fluorescent secondary antibody at 1:200 dilution in 50% blocking buffer for 1 hour at room temperature.
1h
Wash tissue slices in TBS-T(TBS+ 0.25% Triton X-100) for 10 minutes. Repeat x 2.
20m
Wash tissue slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes.
10m
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 .