Feb 05, 2025
In Situ Hybridization -Mendelsohn et al 2025
- Alana Mendelsohn1
- 1Columbia University
Protocol Citation: Alana Mendelsohn 2025. In Situ Hybridization -Mendelsohn et al 2025. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwq5ov8j/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: February 03, 2025
Last Modified: February 05, 2025
Protocol Integer ID: 119508
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 In Situ Hybridization from Mendelsohn et al 2025.
Attachments
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.
Before start
Read all steps and review materials needed before starting this protocol.
Transcardial Perfusion and Tissue Fixation
Transcardial Perfusion and Tissue Fixation
Prepare ice cold 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.
Dissect the brain and place it in OCT (Optimum Cutting Temperature Compound (Tissue-Tek)) in an appropriate mold (avoid bubbles forming).
Rapidly freeze on dry ice then store at -80 °C .
Tissue sectioning
Tissue sectioning
Mount brain onto a cyrostat mount to be able to section in a coronal orientation.
Cut 15μm sections and place sections on Super- Frost Plus Slides, then store at -80 °C .
In Situ Hybridization (ISH)
In Situ Hybridization (ISH)
Fix sections to slides in 4% paraformaldyde (PFA) according to Advanced Cell
Diagonstics (ACD) RNAscope Multiplex Fluorescent V2 Assay manual.
The next steps and visualization steps are detailed in the Advanced Cell Diagonstics (ACD) RNAscope Multiplex Fluorescent V2 Assay manual (in attachments of this protocol).