Mouse anti parvalbumin

Slides were left to thaw to room temperature for 20 min and given three washes in 1X PBS for 10 min each. They were then incubated for 1 h with 10% Protein Block, Serum-Free (Dako, Mississauga, ON) in 1X PBS. After this, slides were incubated overnight at room temperature with a primary antibody in an antibody solution of 1% Protein Block, 1% Bovine Serum Albumin and 98% 1X PBS with 0.1% Triton X-100. The following primary antibodies were used: Wisteria Floribunda Agglutinin (WFA; 1:1000; Vector Labs, Philadelphia, PA), mouse anti-Parvalbumin (1:2000; Swant, Switzerland), rabbit anti-IBA1 (1:200; Dako, Mississauga, ON); and mouse anti-GFAP (1:200; Sigma-Aldrich, Oakville, ON). Slides were washed again three times, twice in 1X PBS with 1% tween-20 and then once in 1X PBS. Sections were then incubated for 1 h at room temperature with secondary antibodies in antibody solution. Secondary antibodies were as follows: Streptavidin 647 (1:200; Invitrogen, Burlington, ON), donkey anti-mouse Alexa Fluor® 488 (1:200; Molecular Probes, Eugene, OR) and donkey anti-rabbit Alexa Fluor® 647 (1:200; Molecular Probes, Eugene, OR). Slides were again washed three times, twice in PBS with 1% Tween-20 and once in 1X PBS. Slides were mounted with DAPI (4′,6-diamidino-2-phenylindole in vectashield mounting medium).

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).