3 3 diaminobenzidine tetrahydrochloride dihydrate

Free-floating brain slices from group I were rinsed in phosphate-buffered solution (PBS), quenched in 3% H₂O₂ and 10% methanol for 15 min (IHC) and blocked in blocking solution (5% normal donkey serum, Jackson ImmunoResearch, UK, and 0.25% Triton X-100 in PBS) for 1 h at room temperature (RT). Sections were incubated overnight at 4 °C with primary antibodies (monoclonal mouse anti-CSPGs, 1:3000 for IHC and 1:1000 for IF, Cat-315/MAB1581, Millipore, USA; goat anti-parvalbumin (PV), 1:2500 for IF, PVG-214, Swant, Switzerland) diluted in blocking solution. Following rinses with 2% normal donkey serum and 0.25% Triton X-100 in PBS, the sections were incubated with appropriate secondary antibodies (donkey anti-mouse for IHC, Vector Laboratories, USA; donkey anti-mouse/goat conjugated with either Cy3 or DyLight 488 fluorescent dyes for IF, Jackson ImmunoResearch, UK) at a dilution of 1:400 in blocking solution for 90 min at RT. IHC visualization was achieved via the Vectastain ABC kit (Vector) using 3,3′-diaminobenzidine-tetrahydrochloride-dihydrate (DabSafe, Saveen Werner, Sweden), 8% NiCl, and 3% H₂O₂. Bright-field pictures were acquired using an Olympus BX60 microscope (Sweden). Fluorescent labelling was imaged using a confocal laser-scanning microscope (Zeiss LSM 510, Germany).