Expression of dlx1a, dlx2a, dlx5a, dlx6a and gad65 was determined using In situ hybridization assays with antisense mRNA probes on crysections as described (Dorsky et al., 1995). Antisense mRNA probes were labeled with digoxygenin-dNTP or dinitrophenol DNP-11-UTP and synthesized from cDNA clones, dlx1a [6 (link)], dlx2a and dlx5a [5 (link)], dlx6a [24 (link)] and gad65 [25 (link)]. Vectors containing the cDNA clones were linearized with BamHI, EcoRI or Xhol and the antisense riboprobes were synthesized using either the T7 or T3 polymerase as required.
Brain sections, stored at -20°C, were thawed at room temperature for 30 minutes before the experiment. Hybridization was carried out overnight at 70°C in a humidified chamber. Slides were washed twice with Solution A (50% Formamide, 5% 20x SSC in dH20) and twice with TBS. Blocking with 10% FBS TBST was carried for 2 hours in RT. Detection of hybridized probes was performed with anti-DIG antibodies AP fragments (Roche, Basel Switzerland; dilution 1:1000) overnight at 4°C. After four TBST washes, staining was developed with NBT/BCIP for 6–18h (Sigma, St-Louis, MO). Images were acquired with a Zeiss AxioPhot Fluorescence Microscope.
Brain sections, stored at -20°C, were thawed at room temperature for 30 minutes before the experiment. Hybridization was carried out overnight at 70°C in a humidified chamber. Slides were washed twice with Solution A (50% Formamide, 5% 20x SSC in dH20) and twice with TBS. Blocking with 10% FBS TBST was carried for 2 hours in RT. Detection of hybridized probes was performed with anti-DIG antibodies AP fragments (Roche, Basel Switzerland; dilution 1:1000) overnight at 4°C. After four TBST washes, staining was developed with NBT/BCIP for 6–18h (Sigma, St-Louis, MO). Images were acquired with a Zeiss AxioPhot Fluorescence Microscope.