Sep 15, 2022
LRRK2 Staining in mouse brain sections
- 1Northwestern university
Protocol Citation: Chuyu Chen 2022. LRRK2 Staining in mouse brain sections . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj64dlmk/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 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70086
Keywords: ASAPCRN
Abstract
LRRK2 is a widely expressed, multifunctional protein.LRRK2 mutations are the major cause to inherited and sporadic PD. Staining of LRRK2 in mouse brain can be challenging. This protocol provides an optimised way to achieve LRRK2 staining in mouse brain sections.
Prepare brain cryosections in 20μm thickness
Blocking with 5% serum (preferably same serum as host of secondary antibody) 30 min RT
Wash with PBS 5 min 2x
Permeabilize with 0.5% Triton 15 min RT
Wash with PBS 5 min 2x
VECTOR® antigen unmasking solution 48ul/5ml water (in small glass sample vial). Microwave 10-20sec until boiled before use (or pre-heat to 100°C on heat plate)
Incubate at 100°C for 30min (<5 section / 5ml) then cool to room temperature (about 20min)
Wash with PBS 5 min 2x
Blocking with 5% serum 16-24hr 4°C
Wash with PBS 5 min 2x
Primary incubation 1% serum + 0.1% Triton 30-48 hr 4°C LRRK2 1:200 (Catalog number: N241A); Co-stained often with TH antibody
Wash with PBS 5 min 2x
Secondary incubation 1% serum + 0.1% Triton 1-2 hr RT Alexa Fluor 1:300
Wash with PBS 5 min 2x
Mount with ProLong™ Gold Antifade Mountant with DAPI