May 09, 2025

Public workspaceConfocal microscopy to assess TelC and iGluSnFR expression in fixed mouse brain slices

DOI
dx.doi.org/10.17504/protocols.io.eq2ly6q4wgx9/v1
  • 1Department of Psychological & Brain Sciences, Boston University, Boston, MA, USA;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
Cláudia C. Mendes
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly6q4wgx9/v1
Protocol Citation: Safa Bouabid, Mark Howe 2025. Confocal microscopy to assess TelC and iGluSnFR expression in fixed mouse brain slices. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6q4wgx9/v1
Manuscript citation:
Bouabid et al., (2025) Distinct spatially organized striatum-wide acetylcholine dynamics for the learning and extinction of Pavlovian associations.
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 05, 2025
Last Modified: May 09, 2025
Protocol Integer ID: 119623
Keywords: mouse, brain, confocal microscopy
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-020370
Abstract
This protocol describes the tissue processing steps following (a) glutamate recordings using the genetically encoded glutamate sensor (iGluSnFR) and (b) behaviour experiments following tetanus toxin light chain (TelC) viral injection in the dorsal medial striatum.

Before start
This protocol was performed in ChAT-IRES-Cre mice (B6;129S6-Chattm2(cre)Lowl/J, JAX stock number 006410).

To block acetylcholine release from cholinergic interneurons, we injected Cre-dependent Tetanus toxin light chain (TelC) viral vector (RRID:Addgene_135391) in the dorsal medial striatum (aDMS) of ChAT-IRES-Cre mice.

To measure rapid changes in glutamate release, we expressed the genetically encoded glutamate sensor iGluSnFR selectively in anterior dorsal striatum (aDS) cholinergic interneurons of ChAT-IRES-Cre mice.
PFA Fixation by Intracardiac Perfusion & Brain Dissection
PFA Fixation by Intracardiac Perfusion & Brain Dissection
Perfuse intracardially with phosphate-buffered saline (PBS 1%, Fisher Scientific), followed by paraformaldehyde (PFA 4% in 1% PBS, Fisher Scientific).
Carefully extract brains and fix them overnight in 4% PFA dissolved in PBS.
Transfer brains to solution of 40% sucrose in 1% PBS (until the brains sank).
Cryosectioning & Mounting
Cryosectioning & Mounting
Slice brains (50μm thickness) with a cryostat (Leica CM3050 S).
Mount coronal sections on super frost slides and cover slipped with Vectashield antifade mounting medium (Vector Laboratories, H-1900).
Image Acquisition using Confocal Microscopy
Image Acquisition using Confocal Microscopy
Acquire images on a Zeiss LSM 800 laser scanning confocal microscope.
Note
TelC and iGluSnFr fluorescence were not immuno-enhanced.