TITLE: Immunocytochemistry of acute brain slices used in ex vivo voltammetry recordings
AUTHORS: Yan-Feng Zhang, Stephanie J Cragg

[DESCRIPTION]
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.

[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. 

[STEPS]
SECTION: Fixation
1. Fix slices in 4% paraformaldehyde (PFA) dissolved in PBS containing 0.2% picric acid.
SECTION: Fixation
2. Leave to fixate overnight at 4°C and then store in PBS. 
SECTION: Fixation
3. Wash x5 in PBS for 5 min. 
SECTION: Labelling Cholinergic Interneurons (ChAT)
7.2. Add primary antibody goat anti-ChAT (Millipore, 1:100) or goat anti-ChAT (1:200, AMCA) to the above mix.
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
14. Prepare PBS containing0.5% Triton X-100, 1% Normal Goat Serum and 1% Foetal Bovine Serum. 
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
14.1. Add secondary antibody DyLight 594 Goat anti-Rabbit (1:1000) or CoraLite488 Goat anti-Rabbit (1:1000) antibody to the above mix.
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
14.2. Once done, remove all the PBS from the last wash and add the Secondary Antibody Solution. 
SECTION: Labelling Cholinergic Interneurons (ChAT)
7.4. 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.
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
13. Secondary Antibody:
SECTION: Confocal Imaging Acquisition
17. 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). 
SECTION: Confocal Imaging Acquisition
18. Maximum intensity projection from a z-stack of height 30 µm was captured individually and the stack of the pictures were compressed.


 

SECTION: Labelling Cholinergic Interneurons (ChAT)
6. This section details steps to label cholinergic interneurons (Chl) expressing channelrhodopsin-2 (ChR2) fused in-frame with eYFP.
SECTION: Pre-incubation
4. Prepare preincubation solution containing PBS with 10% Normal Donkey Serum and 0.5% Triton-X  during the last wash.
SECTION: Pre-incubation
5. Leave to preincubate at room temperature for 1 h on an orbital shaker.
SECTION: Labelling Cholinergic Interneurons (ChAT)
7.3. Once done, remove all the preincubation solution and add the Primary Antibody Solution. 
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
10. 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.
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
11.2. Add primary antibody rabbit anti-TH (1:2000) to the above mix in the desired concentration.
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
11.3. Leave to incubate overnight at 4º C on an orbital shaker.
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
12. Wash x5 with PBS for 5 min.
SECTION: Labelling Cholinergic Interneurons (ChAT)
7. Primary Antibody:
SECTION: Labelling Cholinergic Interneurons (ChAT)
8. Wash x5 with PBS for 5 min.
SECTION: Labelling Cholinergic Interneurons (ChAT)
9. Secondary Antibody:
SECTION: Labelling Cholinergic Interneurons (ChAT)
7.1. Prepare 3% Normal Donkey Serum and 0.5% Triton-X during the preincubation period.
SECTION: Labelling Cholinergic Interneurons (ChAT)
9.1. Prepare PBS containing 0.5% Triton X-100 and 3% Normal Donkey Serum. 
SECTION: Labelling Cholinergic Interneurons (ChAT)
9.2. Add secondary antibody Alexa Fluor 568 donkey anti-goat (1:1000) to the above mix.
SECTION: Labelling Cholinergic Interneurons (ChAT)
9.3. Once done, remove all the PBS from the last wash and add the Secondary Antibody Solution. 
SECTION: Labelling Cholinergic Interneurons (ChAT)
9.4. Leave to incubate for 2h at room temperature on an orbital shaker.
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
11. Primary Antibody:
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
11.1. Prepare 1% Normal Goat Serum, 1% Fetal Bovine Serum and 0.5% Triton-X during the preincubation period.
SECTION: Labelling Dopaminergic neurons and striatal axons (TH)
14.3. Leave to incubate for 2h at room temperature on an orbital shaker.
SECTION: Mounting
15. Wash x5 with PBS for 5 min.
SECTION: Mounting
16. Mount the individual slices on gelled slides with Vectashield mounting medium (Vector Labs).