Rabbit anti chat

The following primary antibodies were used in these experiments: mouse anti- NeuN (Millipore, Etobicoke, Canada; MAB377, 1:1000), Rabbit anti-μ-opioid receptor (Immunostar, Hudson, USA; #24216; 1:8000), mouse anti-parvalbumin (Swant, Fribourg, Switzerland; #235; 1:5000) and rabbit anti-ChAT (Millipore; AB143; 1:600). Sections were removed from antifreeze, rinsed six times in PBS, and then incubated for one hour in a blocking solution (10% bovine serum albumin (BSA), 0.3% Triton-X, 0.1 M PBS, pH 7.4). Next the sections were incubated in primary antibody in PBS containing 0.1% Triton-X and either 2% BSA or 5% NGS for 24–48 h at 4 °C. After washes in PBS, sections were incubated in the following biotinylated secondary antibodies: horse anti-mouse IgG (Vector Laboratories, Burlingame, California, USA; BA-2000; 1:200), goat anti-rabbit IgG (Vector Laboratories; BA-1000; 1:200). Sections were washed once more in PBS and then incubated for 1 h in 1:100 ABC elite kit (PK6100, Vector Laboratories). Antibody binding was revealed using 0.05% 3,3′-diaminobenzidsine (D5905, Sigma-Aldrich, Oakville, ON, Canada) in TBS (pH 7.6) and hydrogen peroxide (0.01%). All slices were then mounted out of distilled water onto slides, counterstained with 0.1% cresyl violet and coverslipped using Permount (SP15, Fisher Scientific).

Cells were fixed with 4% paraformaldehyde for 15 minutes, washed twice with PBS, and then blocked with 0.3% Triton X‐100 containing 10% goat serum for 2 h. After incubation with primary antibodies at 4°C overnight, samples were incubated with the appropriate Alexa Fluor 488 or 594 conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Nuclei were counterstained with Hoechst 33342 (Sigma‐Aldrich; 1:1,000). Primary antibodies included mouse anti‐Nestin (Millipore, Billerica, MA, USA; 1:200), rabbit anti‐Ki67 (Abcam, Cambridge, MA, USA; 1:200), mouse anti‐MAP2 (Millipore; 1:1000), rabbit anti‐GFAP (Millipore; 1:1000), rabbit anti‐ChAT (Millipore; 1:1000) and mouse anti‐Cnp (Millipore; 1:200). Images of stained cells were captured using an EVOS FL Auto (Invitrogen) microscope, and results are expressed as percentages.

Brain sections were pre-treated with 0.3% H₂O₂ in 10 mM PBS (pH 7.4, 4°C) for 30 min. Separate series of sections were respectively incubated at 4°C for 48 h with one of the following primary antibodies: mouse anti-NeuN (1∶500, Millipore), rabbit anti-Darpp32 (1∶200, Cell Signaling, Danvers, MA), mouse anti-Parv (1∶1,000, Sigma), rabbit anti-Cr (1∶2,000, Millipore), rabbit anti-NPY (1∶5,000, ABCAM) and rabbit anti-ChAT (1∶1,000, Millipore). After rinsed in 10 mM PBS for three times (5 min/time), the sections were applied with secondary antibodies anti-mouse IgG or anti-rabbit IgG (both 1∶200, Sigma) at room temperature for 4 h, followed by three rinses (5 min/time) in 10 mM PBS and incubation with homologous peroxidase-antiperoxidase (PAP) complex (1∶200, Sigma) at room temperature for 2 h. The peroxidase reaction was performed using 3, 3′-diaminobenzidine (DAB, 0.05% in 10 mM PBS, pH 7.4, Sigma) for 2–8 min, and then the sections were mounted onto gelatin-coated slides, routinely dehydrated, cleared and covered with neutral balsam for microscopic detection.