Aug 28, 2024
Fluorescent Immunolabelling for endogenous mouse LRRK2
- Roberta Marongiu1,2,
- andrew.west west3,2
- 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;
- 3Duke University
Protocol Citation: Roberta Marongiu, andrew.west west 2024. Fluorescent Immunolabelling for endogenous mouse LRRK2. protocols.io https://dx.doi.org/10.17504/protocols.io.261genqkog47/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: 68930
Keywords: ASAPCRN, LRRK2
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
This protocol is used to label endogenous LRRK2 in mouse.
It has been edited from West et al., 2014
Protocol optimized for fresh fixed brain tissue sectioned at 40 um.
Materials
Primary Antibody anti-LRRK2 c41-2 AbcamCatalog #ab133474
DAPI Thermo Fisher ScientificCatalog #D1306
Donkey serumSigma AldrichCatalog #S30-100ML
Solutions:
Tris Buffer Solution TBS - 2L
- 50mM Tris pH 7.5 – 100 mL of 1M stock
- 150 mM NaCl – 17.53 g
- Distilled Water – 1.9 L
Select brain slices, from fresh, unfrozen, fixed tissue - slices are 40um thick
Wash 3 times for 00:10:00 in TBS
10m
Sections were “quenched” in 100% Methanol for 00:15:00 at 4 °C on shaker.
15m
Wash 3 times for 00:10:00 in TBS
10m
Incubate the slices with Sodium Citrate – 10 mM Sodium Citrate pH 6.0 w/0.05% Tween for 00:30:00 at 37 °C on shaker.
30m
Wash 3 times for 00:10:00 in TBS
10m
During the washes, prepare the blocking solution : 5% normal donkey serum (matched to secondary AB) in TBS with 0.3% Triton
Incubate the slides with blocking solution for 01:00:00 at 4 °C on shaker.
1h
Wash 3 times for 00:10:00 in TBS
10m
During the washes, prepare the primary antibody solution : 5% normal donkey serum in TBS with NO TRITON + Primary Antibody anti-LRRK2 (1:500 or 1:1000)
Incubate at 4 °C on shaker for 24:00:00 48:00:00
3d
Wash 3 times for 00:10:00 in TBS
10m
During the washes, prepare the secondary antibody solution : 5% normal donkey serum in TBS with NO TRITON + Alexa Fluor secondary AB (1:1000)
Incubate at 4 °C on shaker for 18:00:00
18h
Wash 2 times for 00:05:00 in TBS
5m
During the washes, prepare the DAPI solution : DAPI (1mg/ml): 1:10,000, in TBS
Incubate the slides with the DAPI solution for 00:10:00
10m
Wash 3 times for 00:10:00 in TBS
10m
Mount sections on superfrost + slides and coverslip with Invitrogen Prolong Antifade reagent.