Aug 28, 2024

Public workspaceFluorescent Immunolabelling for endogenous ChAT in mice striatum

DOI
dx.doi.org/10.17504/protocols.io.8epv59wk6g1b/v1
  • Roberta Marongiu1,2
  • 1Department of Neurological Surgery, Weill Cornell Medical College, New York, NY 10065;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Jacquelyn Haytayan
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DOI: dx.doi.org/10.17504/protocols.io.8epv59wk6g1b/v1
Protocol Citation: Roberta Marongiu 2024. Fluorescent Immunolabelling for endogenous ChAT in mice striatum. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59wk6g1b/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 19, 2022
Last Modified: September 23, 2024
Protocol Integer ID: 68929
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's Disease
Grant ID: 020608
Abstract
This immunohistochemistry protocol has been designed and adjusted specifically for ChAT immunolabelling in the mice striatum
Materials
ReagentGoat anti-ChAT antibodyMilliporeCatalog #AB144P
ReagentDonkey anti-Goat IgG (H L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 488Thermo Fisher ScientificCatalog #A-11055
ReagentDAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)Thermo FisherCatalog #D1306
ReagentDonkey serumSigma AldrichCatalog #S30-100ML
Solution
Tris Buffer Soaline Triton TBST - 2L
- 50mM Tris pH 7.5 – 100 mL of 1M stock
- 150 mM NaCl – 17.53 g
- Distilled Water – 1.9 L
- Triton 0.2 % - 4 mL

Select floating sections of mouse striatum (30um thick).
Wash: 3 x Duration00:10:00 gently on a shaker, in TBST at TemperatureRoom temperature .

10m
During the washes, prepare blocking solution: 10 % donkey serum (DS) in TBST.
Blocking step: Incubate the slides with blocking solution on the shaker for Duration01:00:00 at TemperatureRoom temperature .
1h
Before the end of the blocking step, prepare the solution with the primary antibody ChAT
blocking solution (TBST+10% DS) + Goat Anti-Chat Ab dilution 1:200.
Primary antibody: incubate the slides with the blocking solution/Antibody on a shaker at Temperature4 °C for Duration24:00:00 Duration48:00:00 .

3d
Washes: 3 x Duration00:10:00 on a shaker, in TBST.

10m
During the washes, prepare the secondary Antibody solution
blocking solution (TBST+10% DS) + anti donkey anti goat Alexa Fluor 1:1000.
incubate the slides with Secondary antibody solution Duration01:00:00 at TemperatureRoom temperature .

1h
Washes: 2 times, for Duration00:10:00 in TBST.

10m
Optional: DAPI staining
During the washes, prepare the DAPI solution
DAPI (1 mg/ml). Dilution 1:10.000 in TBST.
Incubation the slides Duration00:10:00 with the DAPI solution.

10m
Washes: 2 times, for Duration00:10:00 each in TBST.

10m
Mounting on Superfrost+ slides.
Coverslip: mounted with prolong anti-fade reagent (Invitrogen).