Feb 05, 2025
Immunohistochemistry (Histology + Imaging)-Mendelsohn et al 2025
- Alana Mendelsohn1
- 1Columbia University
Protocol Citation: Alana Mendelsohn 2025. Immunohistochemistry (Histology + Imaging)-Mendelsohn et al 2025. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwqywv8j/v1
Manuscript citation:
Segregated basal ganglia output pathways correspond to genetically divergent neuronal subclasses
Alana I. Mendelsohn, Laudan Nikoobakht, Jay B. Bikoff, Rui M. Costa
bioRxiv 2024.08.28.610136; doi: https://doi.org/10.1101/2024.08.28.610136
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: January 31, 2025
Last Modified: February 05, 2025
Protocol Integer ID: 119408
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020551
Abstract
This protocol outlines the steps for preparing mouse brain tissue for immunostaining and imaging from Mendelsohn et al 2025.
Safety warnings
Wear appropriate PPE as required by your institution.
Ethics statement
Prior ethics approval (e.g. IACUC) should be obtained before performing these experiments. Approval was obtained by the Columbia University IACUC before any procedures were performed.
Transcardial Perfusion and Tissue Fixation
Transcardial Perfusion and Tissue Fixation
Prepare PBS and ice cold 4% PFA (paraformaldehyde) in PBS for perfusion.
Set up the perfusion pump and fill the tubing with PBS.
Deeply anesthetize the mouse using isoflurane.
Pin the mouse to a Styrofoam plate.
Make a midline incision to expose the thoracic cavity, cutting through the skin and rib cage.
Insert the needle into the left ventricular chamber of the heart.
Switch on the perfusion pump and open the right atrium using sharp forceps immediately.
Perfuse mouse with PBS to flush out the blood.
Switch perfusion pump to the ice cold 4% PFA.
Dissect the brain and place it in 4% PFA for post-fixation overnight at 4 °C .
Wash brain x3 in PBS.
Transfer the brain to a 30% sucrose solution in PBS at 4 °C for 3 days.
Brains are embedded in OCT (Optimum Cutting Temperature Compound (Tissue-Tek)) then stored at -80 °C .
Tissue sectioning
Tissue sectioning
Mount brain onto a cyrostat mount using OCT to be able to section in a coronal orientation.
Cut 50 μm sections and place sections in 24 well plates with PBS.
Staining (IHC)
Staining (IHC)
2d 2h 15m
2d 2h 15m
NOTE-stain sections in 24 well plates.
Dilute primary antibodies in PBST 0.3%, 0.5% BSA and 0.05% thimersol.
Primary Antibodies and concentrations are:
Rabbit anti-Foxp2 (Abcam, 1:10k)
Guinea Pig anti-Pou6f2 (Columbia, 1:2k)
Rabbit anti-GFP-488 (Invitrogen, 1:1k)
Chicken anti-GFP (Aves labs, 1:2k)
Rabbit anti-RFP (Rockland, 1:2k)
Rat anti-mCherry(Invitrogen, 1:2k)
Mouse anti-TH (Immunostar, 1:10k)
Chicken anti-Myc (Invitrogen, 1:250)
Incubate tissue sections with primary antibodies at 4 °C for 48:00:00 .
2d
Wash sections 3 times in PBS.
Incubate sections with secondary antibodies at a 1:1000 dilution for 02:00:00 at Room temperature .
Use the below secondary antibodies pending primary antibodies used in sections.
Goat anti-Chicken 488 (Invitrogen)
Goat anti-Guinea Pig 568 (Invitrogen)
Goat anti-Mouse 488 (Invitrogen)
Goat anti-Mouse 568 (Invitrogen)
Goat anti-Rabbit 568 (Invitrogen)
Goat anti-Rabbit 647 (Invitrogen)
Goat anti-Rat 568 (Invitrogen)
2h
Wash sections 3 times in PBS.
Stain for DAPI (1:1000 in PBS) at Room temperature for 00:15:00 .
15m
Mount sections onto Super- Frost Plus Slides with coverslips and desired mounting media.
Imaging
Imaging
Pending experimental need visualize sections using:
A W1- Yokogawa inverted spinning disk confocal using a 10x or 20x objective.
OR
Automated high-throughput imaging of tissue sections was performed on a custom-built
automated slide scanner using a AZ100 microscope equipped with a 4x 0.4NA Plan Apo objective
(Nikon Instruments Inc) and P200 slide loader (Prior Scientific), controlled by NIS-Elements using
custom acquisition scripts (Nikon Instruments Inc.).