Mar 23, 2026

Public workspaceImmunohistology to confirm ChR2 expression in post-mortem mouse brain slices

 Forked from Immunohistology
DOI
https://dx.doi.org/10.17504/protocols.io.6qpvrbqmolmk/v1
Immunohistology to confirm ChR2 expression in post-mortem mouse brain slices
  • Mai-Anh Vu1,2,
  • Mark Howe1,2
  • 1Department of Psychological and Brain Sciences, Boston University, Boston, MA, United States;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
Mai-Anh Vu
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvrbqmolmk/v1
Protocol Citation: Mai-Anh Vu, Mark Howe 2026. Immunohistology to confirm ChR2 expression in post-mortem mouse brain slices. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrbqmolmk/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: March 10, 2026
Last Modified: March 23, 2026
Protocol Integer ID: 313034
Keywords: stain for neuron, neuron, moving mice, immunohistology step, micro, neurotransmitter, investigation of cell, fiber array, immunohistology, mice, viral expression, immunohistology step, mouse striatum, immunohistology, chr2 expression, chr2, viral expression
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020370
Abstract
This protocol includes the immunohistology steps we used to confirm channelrhodopsin-2 (ChR2) viral expression after our micro-CT scanning and contrast agent soaking procedure (see Protocol: Micro-CT scanning for post-implant localization of multi-fiber arrays in mouse striatum).


Materials
Reagents:
  • Euthasol (Virbac, Cat#710101)
  • Normal Goat Serum (Thermo Fisher Scientific Cat# 31873)
  • Phosphate Buffered Saline (ThermoFisher, Cat# BP39920
  • Sodium Thiosulfate, Anhydrous (Carolina Sciences, Cat#892020)
  • Triton X-100 (Sigma-Aldrich, Cat# 93443)
  • Vectashield Mounting Medium (H-1900) (Vector Laboratories, Cat# 10262)

Antibodies:

Equipment:
Troubleshooting
Removing Lugol's contrast agent from brain tissue
In preparation for immunohistology, Lugol’s solution was removed from brain tissue after CT imaging by soaking the implanted brains in a solution of 5% sodium thiosulfate (STS) (see Materials) in 1% PBS for 4-6 days (Hopkins et al., 2015).
The implant was then removed, the brain returned to the STS solution for one hour.

Slicing
The brains were then moved to a solution of 30-40% sucrose in PBS.
Once the brains sank, coronal sections (50 µm) were sliced with a cryostat (Leica CM3050 S) and transferred to PBS for storage.
Immunostaining
To stain for ChR2 expression:

Sections were then blocked and treated in a 5% normal goat serum (NGS) and 0.2% PBS Triton solution (PBST).
Treated sections were then incubated for 24-48 hours at 4°C in 5% NGS, 0.2% triton, and a GFP primary antibody (1:1000) (see Materials).
Slices were then washed, and incubated for 2 hours at room temperature in 5% NGS, 0.2% PBST, and an Alexa™ 488 secondary antibody (1:200) (see Materials).
Mounting and imaging
Finally, stained sections were mounted onto glass slides using Vectashield Mounting Medium (see Materials).
Confocal images were acquired with an Olympus VS120 slide scanner.
Protocol references
Hopkins, T.M., Heilman, A.M., Liggett, J.A., LaSance, K., Little, K.J., Hom, D.B., Minteer, D.M., Marra, K.G., and Pixley, S.K. (2015). Combining micro-computed tomography with histology to analyze biomedical implants for peripheral nerve repair. J. Neurosci. Methods 255, 122–130.