Mms 435p

Cells were fixed in 4% (v/v) paraformaldehyde and washed with PBS. Samples were permeabilized in 0.1% (v/v) Triton-X 100 in PBS for 15 min at room temperature, washed in PBS and blocked in 5% (w/v) BSA for 30 min at room temperature. Primary antibodies were diluted in 1% (w/v) BSA, incubated overnight at +4°C and were the following:
mouse anti-GFAP monoclonal antibody MAB360 (Millipore);
mouse anti-neuronal class IIIb-tubulin (TUJ1) monoclonal antibody MMS-435P (Covance).
Samples were washed in PBS and incubated in Alexa Fluor 555 goat anti-mouse (Life Technologies) together with Hoechst nuclear dye for 20 min at room temperature. After further washes in PBS, slides were mounted in PBS/glycerol and analysed under a fluorescence microscope. Images were analysed and scored using ImageJ.

Cultures were fixed in freshly prepared, buffered 4% paraformaldehyde. After blocking with 10% normal goat serum (NGS), cultures were incubated overnight at 4 °C with the following antibodies (mAbs, monoclonal; pAbs, polyclonal): β-tubulin isotypeIII (β-tubIII, mAbs, MMS-435 P (Covance, Princeton, NJ, USA), 1 : 400), GFAP (pAbs (Dako, Cernusco sul Naviglio, Milan, Italy), 1 : 400), Lamp1 (pAbs, ab24170 (Abcam, Cambridge, UK), 1 : 750), cleaved caspase-3 (Casp; Asp 170) (pAbs, no. 9961 (Cell Signaling Technology, Danvers MA, USA), 1 : 500), Ub (pAbs, Z0458 (Dako), 1 : 50) MAP2 (mAbs (Chemicon, EMD Millipore, Darmstadt, Germany), 1 : 400).
After removal of the primary Abs and repeated washes with Dulbecco's phosphate-buffered saline (PBS), cultures were incubated at room temperature for 45 min with secondary antibodies labeled with Alexa Fluor 594 or 488 (anti-mouse and/or anti rabbit; Molecular Probes, ThermoFisher Scientific, Waltham, MA, USA). Samples were then colored with DAPI (4′,6-diamidino-2-phenylindole; 0.3 μg/ml; Roche, Basel, Switzerland) for nuclear staining and rinsed with PBS for mounting and analysis. Microphotographs were taken using a Zeiss Axiovert 200 direct epifluorescence microscope (Axioplan 2; Carl Zeiss, Jena, Germany) or a confocal microscopy (Leica DM IRE2, Milan, Italy).

E12.5 cortices from Neurog1^+/−; Neurog2^GFP/+ heterozygous intercrosses were dissociated using trypsin, FACS sorted into GFP⁺ and GFP⁻ populations and plated at 200 cells per well on a feeder layer of rat cortical cells in neurosphere media containing bFGF (2 ng/ml) for 7 days. Cells were stained using M2/M6 (DSHB) to distinguish mouse cells from rat cells. Clones were stained with anti-Tuj1 (neuronal III β-tubulin, 1/500, Covance, MMS-435P) and quantified as containing only neurons, no neurons or a mix of neurons and other cell types. Clone size was also assessed.

The following primary antibodies were used: anti-BIG1 N-terminal (Santa Cruz, sc-376790), anti-BIG1 C-terminal (Bethyl, A300-998A), anti-Lamin-B (Santa Cruz, sc-6216) anti-Pax6 (Covance, PRB-278P), anti-cleaved-Caspase-3 (Cell Signaling, #9661), anti-Calbindin (Santa Cruz, sc-7691), anti-Prox1 (R&D Systems, AF2727), anti-Ctip2 (Abcam, ab18465, gift from Dr. Makoto Sato, Osaka University), anti-Tbr1 (Chemicon, AB9616; Santa Cruz, sc-15607), anti-Cux1 (Santa Cruz, sc-13024), anti-BrdU (BD Pharmingen, 555627), anti-Map1b (clone 1B6, gift from Dr. Reiko Harada, Takarazuka University of Medical and Health Care) [20 (link)], anti-Tau1 (Chemicon, LV1383492), anti-Map2 (Cell Signaling, #4542), anti-Tuj1 (Covance, MMS-435P) and anti-Golgin97 (Gift from Dr. Nobuhiro Nakamura, Kyoto Sangyo University) [21 (link)]. The following secondary antibodies were used: HRP-conjugated goat anti-mouse (ICN Pharmaceuticals, 59296), calf anti-rabbit (Rockland, 200–4135) or calf anti-goat (Rockland, 200–1135), Alexa-Fluor-488-labeled or Alexa-Fluor-594-labeled donkey anti-mouse (Invitrogen, A21202; Molecular Probes, A21203), donkey anti-rabbit (Invitrogen, A21206; Molecular Probes, A21207), donkey anti-rat (Molecular Probes, A21208) and donkey anti-goat (Molecular Probes, A11055).