Apr 02, 2025
Immunohistochemistry & Microscopy-Falasconi & Kanodia et al 2025
- Antonio Falasconi1,2,3,
- Harsh Kanodia1,2,3,
- Silvia Arber1,2,3
- 1Biozentrum, University of Basel;
- 2Friedrich Miescher Institute for Biomedical Research;
- 3Aligning Science Across Parkinson’s (ASAP)
Protocol Citation: Antonio Falasconi, Harsh Kanodia, Silvia Arber 2025. Immunohistochemistry & Microscopy-Falasconi & Kanodia et al 2025. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7m7k9gwz/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: February 18, 2025
Last Modified: April 02, 2025
Protocol Integer ID: 120855
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020551
European Union’s Horizon 2020 research and innovation programme
Grant ID: InterAct, grant agreement No 101018151
Swiss National Science Foundation
Grant ID: N/A
Abstract
Immunohistochemistry and microscopy methods used in Falasconi & Kanodia et al 2025.
Guidelines
Anesthesia:
Animals are anesthetized with a ketamine–xylazine solution.
Materials
Antibodies:
| A | B | C | D | |
| Chicken anti-GFP | Invitrogen | RRID: AB_2534023 | Cat# A10262 | |
| Rabbit anti-RFP | Rockland | RRID: AB_2209751 | Cat# 600-401-379 | |
| chicken anti-TH | Neuromics | RRID: AB_2201403 | Cat# CH23006 | |
| goat anti-ChAT | Millipore | RRID: AB_2079751 | Cat# AB144P | |
| Donkey anti-rabbit Cy3 | Jackson Immuno Research | RRID: AB_2307443 | Cat#711-165-152 | |
| Donkey anti-goat Cy5 | Invitrogen | RRID: AB_2535864 | Cat# A-21447 | |
| Donkey anti-chicken 488 | Jackson Immuno Research | RRID: AB_2340375 | Cat#703-545-155 | |
| Donkey anti-chicken Cy5 | Jackson Immuno Research | RRID: AB_2340379 | Cat#703-605-155 | |
| Donkey anti-goat 488 | Invitrogen | RRID: AB_2534102 | Cat# A-11055 |
Safety warnings
Wear appropriate PPE as required by your institution.
Ethics statement
Prior ethics approval should be obtained before performing these experiments. Approval was obtained from the Cantonal Veterinary Office Basel-Stadt and performed in compliance with the Swiss Veterinary Law guidelines.
Before start
Read through the entire protocol before starting.
Transcardial Perfusion and Tissue Fixation
Transcardial Perfusion and Tissue Fixation
Prepare PBS and 4% PFA in PBS for perfusion.
Set up the perfusion pump and fill the tubing with PBS.
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 or small scissors immediately.
Perfuse the mouse with PBS to flush out the blood.
Switch the perfusion to 4% PFA and perfuse until limbs are stiff.
Dissect the brain and spinal cord then place it in 10 mL of 4% PFA for post-fixation overnight at 4°C.
Transfer the brain to a 30% sucrose solution in PBS at 4°C until the tissue sinks (24–48 hours).
Embed the brain in OCT compound and freeze at -80°C.
Sectioning
Sectioning
Mount brain onto a cyrostat mount using OCT to be able to section in a coronal orientation.
Cut 80 μm sections and place sections in 24 well plates with PBS
Staining (IHC)
Staining (IHC)
Incubate floating sections for 1 hour in blocking solution (1% BSA, 0.2% Triton X-100, PBS) at room temperature.
Add primary antibodies to the blocking solution and incubate for 1-3 days at 4°C.
Floating sections are washed x3 with PBS for 10 minutes each wash.
Add fluorophore-coupled secondary antibodies in blocking solution and incubate for 1 day at 4°C.
Floating sections are washed x3 with PBS for 10 minutes each wash.
Mount sections using fluorescence-compatible mounting medium and carefully place coverslips.
Imaging
Imaging
For low-resolution overview imaging: scan slides with an Axioscan light microscope (Zeiss).
For higher resolution imaging use an Axio Imager M2 microscope (Zeiss) with a Yokogawa CSU W1 Dual camera T2 spinning disk confocal scanning unit.