Nov 12, 2023
Immunohistochemistry
- 1University of California, San Diego
Protocol Citation: Leonardo A Parra-Rivas 2023. Immunohistochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xmzdlqe/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: November 12, 2023
Last Modified: November 12, 2023
Protocol Integer ID: 90824
Abstract
Immunohistochemistry
Mice were euthanized using isoflurane (VetOne Cat#502017) inhalation followed by
cervical dislocation. At autopsy, mouse brains were removed from the calvarium
and rapidly placed in a 4% paraformaldehyde (PFA) solution (Thermo Scientific,
Cat# J19943-K2) for drop fixation.
For immunohistochemistry, 5 mm sagittal tissue sections were stained with antibodies against total a-syn (Synaptic Systems, 1:15,000, Cat #128211) and Ser129P a-syn (Cell Signaling, 1:3000, Cat #23706). Slides were stained using Ventana Discovery Ultra (Ventana Medical Systems, Tucson, AZ, USA).
The sections were deparaffinized by three cycles of heating to 72°C in the presence of Discovery Wash (Ventana, Ref# 07311079001) for 4 min with a rinse between each heating step.
Subsequently, antigen retrieval was performed using CC1 buffer (Tris-EDTA; pH
8.6 Ventana, Ref# 06414575001) for 40 min (5 cycles of 8 min each with fresh
CC1 buffer for each incubation) at 100°C. Endogenous peroxidase was quenched by
incubation with inhibitor CM (Ventana, Cat# 760-159) for 8 min. The sections
were incubated with primary antibodies for 32 minutes at 37 °C, mixing the samples every 4 min to
ensure an even distribution of the antibody.
Primary antibodies were detected
using the OmniMap system, with an anti-rabbit secondary antibody coupled to an
HRP polymer (Roche Cat# 760-4311, RRID:AB_2811043) (Ventana, Ref# 05266548001).
Antibody presence was visualized using the Ventana Discovery ChromoMap DAB kit
(Ventana, Ref# 05266645001) as a chromagen followed by hematoxylin II (Ventana,
Ref#05277965001) for 4 min followed by a 4 min treatment with bluing reagent
(Ventana, Ref# 05266769001) as a counterstain.
The slides were rinsed,
dehydrated with alcohol and xylene, and mounted on coverslips. To confirm the
specificity of the pSer129 antibody, the epitope was dephosphorylated using
Lambda Protein Phosphatase (New England Biolab, Cat # P0753). Briefly, 2400
units of the Lambda Protein Phosphatase were supplemented with a 10x NEBuffer
and 10x MnCl2 and added to the slides for 2h at 37°C. Antigen retrieval and peroxidase
quenching were performed on the slides before incubation with the pSer129
antibody.
For quantification of Ser129P staining, after background correction,
the following ROIs were placed to sample the frontal lobe and hippocampus – ROI
measuring ~ 2.5 mm x 1 mm flanking the frontal pole, and an irregular ROI over
the CA3 region (which was strongly positive for Ser129P and easy to identify).
Average intensities were considered for quantification, and data were analyzed
in GraphPad Prism software [(RRID:SCR_002798) http://www.graphpad.com/].