Mouse monoclonal anti smi 32 antibody

Mice were anesthetized with an overdose of rodent cocktail (a combination of ketamine 50 mg/kg, xylazine 5mg/kg, acepromazine 1mg/kg injected i.p.) and perfused transcardially with 0.9% saline, followed by 4% PFA in PBS (pH 7.4). Brains were immersed in 30% sucrose at 4°C until sunk and flash frozen with cold 2-methylbutane (Fisher Scientific, Hampton, NH, USA). Coronal sections of the pregenual mPFC were obtained at 35 μm using a cryostat (Leica CM3050 S, Concord, ON, Canada). For double-labeled immunofluorescence, endogenous mouse antibodies were blocked using a mouse Ig blocking reagent (Vector Laboratories, Burlingame, USA). mPFC sections were incubated overnight at 4°C with mouse monoclonal anti-SMI-32 antibody (1:1000 dilution, Covance, Burlington, NC, USA, Cat. #14941802) and Goat anti-GFP antibody (1:1000 dilution, Novus Biologicals, Oakville, ON, Canada, Cat. #NB100–1770). Immunostaining was visualized with Alexa 488-conjugated (Jackson Immunoresearch, Van Allen Way, Carlsbad, CA, USA, Cat. #705–545-003), and Alexa Fluor 555-conjugated (Life technologies, Toronto, ON, Canada, Cat. #A21429) secondary antibodies raised in donkey.