Rabbit anti numb

Cells were allowed to settle on poly-L-lysine coated coverslips (BD Biosciences) at 37°C, fixed with 4% paraformaldehyde (USB Corporation) or methanol, permeabilized with 1X Dako wash buffer (Dako) and blocked with 20% normal goat serum (Invitrogen) or donkey serum (Abcam) in 1X Dako wash buffer. Primary antibody incubation was overnight at 4°C. The following primary antibodies were used: rabbit anti-Numb 1:50 or 1:100 (Abcam), goat anti-LIS1 1:500 (Santa Cruz Biotechnology), mouse anti-alpha-tubulin 1:200 (Abcam), rat anti-α-tubulin 1:1000 (Abcam), mouse anti-α-tubulin conjugate FITC 1:200 (Sigma). Secondary antibody incubation was performed for 1 hr at room temperature. DAPI (Molecular Probes) was used to detect DNA. Images were obtained with a Confocal Leica TCS SP5 II (Leica Microsystems) or an Axio Observer.Z1 microscope with the LSM 700 scanning module (Zeiss). ImageJ 1.46r was used to determine fluorescence intensity.

We used the click-it® chemical reaction protocol for EdU detection followed by immunostaining with primary and secondary antibodies and before DAPI staining. For confocal microscopy, embryos were fixed in 4% paraformaldehyde overnight and then 30% sucrose, and then embedded in OCT, sectioned and stained using standard protocols. Antibodies used were: goat and rabbit anti-β1-integrin (1:400; R&D or 1:1000; Abcam), mouse anti-Isl1 (1:200, Iowa Hybridoma Bank), rabbit anti-RFP (1:400, Clontech), rabbit anti-Numb (pre-absorbed, 1: 500, Abcam or from Dr. Zhong), and mouse anti-PH3 (1:500, Abcam). Alexa Fluor secondary antibodies (Invitrogen) were used for all secondary detection and confocal images acquired with a Zeiss LSM 510 Meta confocal microscope using Zen™ acquisition software. For Western blotting, cell lysate was resolved on SDS-PAGE and electroblotted on nitrocellulose membranes and incubated with primary antibodies in 5% nonfat milk overnight at 4 degrees Celsius. Secondary antibodies were incubated for 1 hour at room temperature. The blots were washed 3×10 mins in TTBS, and detection was by chemiluminescence (Amersham ECL, GE Healthcare Life Sciences).