Sep 09, 2024
ChAT and human alpha-synuclein immunofluorescence staining
- 1German Center for Neurodegenerative Diseases (DZNE)
Protocol Citation: Pietro La Vitola 2024. ChAT and human alpha-synuclein immunofluorescence staining. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49y2jgo5/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 09, 2024
Last Modified: September 09, 2024
Protocol Integer ID: 107165
Keywords: choline acetyltransferase, ChAT, immunofluorescence, human alpha-synuclein
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol is designed for detecting choline acetyltransferase (ChAT) using the Millipore AB144P (RRID:AB_2079751) and human alpha-synuclein (Abcam, RRID:AB_2537217, MJFR1). Tissues stained with this protocol include 35 μm free-floating mouse brain sections. All tissues were from mice perfused with 4% PFA.
ChAT and human alpha-synuclein immunofluorescence staining
ChAT and human alpha-synuclein immunofluorescence staining
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 and anti-hASYN (1:10000; RRID:AB_2537217, MJFR1 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 .