NFIX expression in brain sections was analyzed using a rabbit polyclonal anti-NFIX serum raised against amino acids 277–291 of human NFIX made by Geneka Biotechnology Inc (cat# 16021118, Montreal, Canada) and now available from Active Motif (cat# 39072, Carlsbad, CA, USA). Other antibodies used were as follows: Pax6 (Chemicon, rabbit), doublecortin (DCX, Santa Cruz sc-8606, goat) and phospho-histone H3 (pH3, Upstate, rabbit). Sections were washed with 1X phosphate buffered saline (PBS) and incubated for 2 hours with a blocking buffer containing 2% goat serum (v/v, S-1000, Vector Laboratories, Burlingame, CA) and 0.2% Triton X-100 (v/v; Sigma, St. Louis, MO) in 1X PBS. Anti-NFIX was used at 1/20,000 for embryonic tissues and 1/12,500 for postnatal tissues, anti-Pax6 was used at 1/25,000, anti-DCX was used at 1/500 and anti-pH3 was used at 1/2000. Sections were incubated overnight at room temperature then washed several times in PBS and incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) for 1 to 2 hours at room temperature, followed by processing with a VECTASTAIN ABC kit for 1 hour at room temperature (A used at 1/500, B used at 1/500, PK6100, Vector Laboratories). Sections were processed for color reaction using nickel- 3,3'-Diaminobenzidine (DAB, D5905, Sigma) solution (2.5% nickel sulfate and 0.02% DAB in 0.175 M sodium acetate) activated with 0.01% (v/v) hydrogen peroxide or the glucose-oxidase modification thereof [36 (link)], washed with 1X PBS multiple times, mounted on 3% gelatin-coated slides and coverslipped with DPX mounting medium (Electron Microscopy Sciences, PA). Immunofluorescent staining was performed using a Cy3-labeled goat anti-rabbit secondary antibody and DAPI (300 nM) staining of nuclei. Images were acquired on a PowerPhase digital camera (PhaseOne, Coppenhagen, Denmark), Zeiss AxioCam HRc (Zeiss, Germany) or a Zeiss LSM 510 Meta confocal microscope and were processed using Adobe Photoshop.
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