Jan 30, 2025
Immunocytochemistry of acute brain slices used in ex vivo voltammetry recordings
- Yan-Feng Zhang1,2,
- Stephanie J Cragg3,2,4
- 1Department of Clinical and Biomedical Sciences, University of Exeter, Exeter, United Kingdom;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 3Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK;
- 4Oxford Parkinson’s Disease Centre, University of Oxford, Oxford, United Kingdom
Protocol Citation: Yan-Feng Zhang, Stephanie J Cragg 2025. Immunocytochemistry of acute brain slices used in ex vivo voltammetry recordings. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3jw5lk5/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: November 28, 2023
Last Modified: January 30, 2025
Protocol Integer ID: 91487
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020370
Parkinson's UK
Grant ID: J-1403; G-1305
Medical Research Council
Grant ID: MR/K013866/1, MR/J004324/1
Abstract
The steps detailed in this protocol are to label choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH), which are known markers for cholinergic interneurons (Chl) and dopaminergic neurons and striatal axons (DA), respectively. We use an immunofluorescent approach in 300-µm thick slices of mouse brain tissue after performing ex vivo voltammetry recordings.
Materials
Equipment:
- MS 3.1 digital shaker (IKA, SKU #0003319000)
- Pastuer pipette (VWR, SKU #612-1681)
Reagents:
- Picric Acid
- Phosophate buffered saline (Sigma-Aldrich, SKU #P4417)
- Phosphate buffer: Sodium phosphate dibasic dihydrate (0.2M) (Sigma-Aldrich, SKU #71643, CAS #10028-24-7) & Sodium phosphate monobasic monohydrate (0.2M) (Sigma-Aldrich, SKU #S9638, CAS #10049-21-5)
- Triton-X 100 (Sigma-Aldrich, X100, CAS #9036-19-5)
- Vectashield (Vector Labs, SKU #H-1900-10)
- Goat anti-ChAT primary antibody (Millipore, SKU #AB114P, RRID #AB_2313845)
- Goat anti-ChAT primary antibody (AMCA, Jackson Immuno Research Europe)
- Rabbit anti-TH primary antibody (Sigma-Aldrich)
- Alexa Fluor 568 Donkey anti-Goat antibody (Invitrogen)
- CoraLite488 Goat anti-Rabbit antibody (Proteintech)
Before start
We first performed voltammetry recordings in ex vivo mouse brain slices following the steps detailed in Protocol: Fast-scan cyclic voltammetry to assess dopamine release in ex vivo mouse brain slices.
Fixation
Fixation
Fix slices in 4% paraformaldehyde (PFA) dissolved in PBS containing 0.2% picric acid.
Leave to fixate overnight at 4°C and then store in PBS.
Wash x5 in PBS for 5 min.
Pre-incubation
Pre-incubation
Prepare preincubation solution containing PBS with 10% Normal Donkey Serum and 0.5% Triton-X during the last wash.
Leave to preincubate at room temperature for 1 h on an orbital shaker.
Labelling Cholinergic Interneurons (ChAT)
Labelling Cholinergic Interneurons (ChAT)
This section details steps to label cholinergic interneurons (Chl) expressing channelrhodopsin-2 (ChR2) fused in-frame with eYFP.
Primary Antibody:
Prepare 3% Normal Donkey Serum and 0.5% Triton-X during the preincubation period.
Add primary antibody goat anti-ChAT (Millipore, 1:100) or goat anti-ChAT (1:200, AMCA) to the above mix.
Once done, remove all the preincubation solution and add the Primary Antibody Solution.
Leave to incubate overnight (goat anti-ChAT from Millipore) or for five days (goat anti-ChAT from AMCA) at 4º C on an orbital shaker.
Wash x5 with PBS for 5 min.
Secondary Antibody:
Prepare PBS containing 0.5% Triton X-100 and 3% Normal Donkey Serum.
Add secondary antibody Alexa Fluor 568 donkey anti-goat (1:1000) to the above mix.
Once done, remove all the PBS from the last wash and add the Secondary Antibody Solution.
Leave to incubate for 2h at room temperature on an orbital shaker.
Labelling Dopaminergic neurons and striatal axons (TH)
Labelling Dopaminergic neurons and striatal axons (TH)
This section details steps to label dopaminergic neurons and striatal axons (DA) expressing (i) channelrhodopsin-2 (ChR2) fused in-frame with eYFP; (ii) genetically encoded calcium indicator GCaMP6f; (iii) voltage sensor ASAP3 without a soma-targeting signal; or (iv) optogenetic actuator Chrimson.
Primary Antibody:
Prepare 1% Normal Goat Serum, 1% Fetal Bovine Serum and 0.5% Triton-X during the preincubation period.
Add primary antibody rabbit anti-TH (1:2000) to the above mix in the desired concentration.
Leave to incubate overnight at 4º C on an orbital shaker.
Wash x5 with PBS for 5 min.
Secondary Antibody:
Prepare PBS containing0.5% Triton X-100, 1% Normal Goat Serum and 1% Foetal Bovine Serum.
Add secondary antibody DyLight 594 Goat anti-Rabbit (1:1000) or CoraLite488 Goat anti-Rabbit (1:1000) antibody to the above mix.
Once done, remove all the PBS from the last wash and add the Secondary Antibody Solution.
Leave to incubate for 2h at room temperature on an orbital shaker.
Mounting
Mounting
Wash x5 with PBS for 5 min.
Mount the individual slices on gelled slides with Vectashield mounting medium (Vector Labs).
Confocal Imaging Acquisition
Confocal Imaging Acquisition
Acquire images using a 20×/0.75 NA objective in Zeiss LSM880 confocal microscope system running ZEN black version 2.3 (Zeiss), or a confocal microscope system (FV1000 IX81; Olympus) and Fluoview software (Olympus).
Maximum intensity projection from a z-stack of height 30 µm was captured individually and the stack of the pictures were compressed.
Note
Red fluorescence (TH and ChAT) was captured at 638–759 nm with 633 nm excitation.
Green fluorescence (GCaMP, ChR2, and ASAP3) was captured at 493–630 nm with 488 nm excitation.