Rabbit anti numb

To detect Gli1 and Numb, neurospheres were blandly disaggregated and plated on poly-lysine-coated Lab-Tek chamber slides (cover slips) for 2 h. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, incubated in blocking solution (5% normal goat serum, 1% BSA, 0.1% Triton X-100) and stained overnight with primary antibodies diluted in blocking solution and for 2 h with secondary antibodies. Primary antibodies were mouse anti-Gli1 and rabbit anti-Numb (Cell Signaling Technology Inc). 594- or 488-conjugated anti-mouse and anti- rabbit secondary antibodies were purchased from Molecular Probes (Invitrogen, Eugene, OR). Nuclei were counterstained with Hoechst reagent. Cover slips were mounted with fluorescence mounting medium (Dako, Carpinteria, CA). Images were acquired with Carl Zeiss microscope (Axio Observer Z1) and AxioVision Digital Image Processing Software.

Transmission electron microscopy was performed as previously described (36 (link)). Hematoxylin & eosin staining was carried out as described (37 (link)). For immunostaining, embryos or tissues were fixed with 4% paraformaldehyde overnight at 4°C and then rinsed with PBS 3 times. The samples were dehydrated in an increasing gradient of 10% to 30% sucrose/PBS and embedded in OCT (Tissue Tek). The samples were cryosectioned at 10-μm thickness, and OCT was washed out by PBS. The sections were incubated with diluted primary antibody overnight at 4°C and then with cy2- or cy3-labeled secondary antibody (Jackson Immunoresearch) for 1 hour at room temperature with PBS wash 3 times in between. The following antibodies were used for immunostaining: rabbit-anti-Numb (Cell Signaling, 2756s, 1:200 dilution), mouse anti-α-Actinin (Sigma, A7811, 1:500 dilution), mouse anti-α-Actin (Sigma, A9357, 1:500 dilution), goat anti-NEBL (Abcam,ab99420, 1:100 dilution), mouse anti-α-Tubulin (Covance, MMS-407R, 1:800 dilution), mouse anti-Desmin (Covance, MMS-454s, 1:200 dilution), and Phalloidin (Invitrogen, A12379 1:500 dilution).