Rabbit anti n1icd

Embryos were fixed in 4% paraformaldehyde for 2 h or overnight at 4 °C, cryopreserved in 20% sucrose, and embedded in OCT compound for cryosectioning. Sections were blocked in 10% Dako serum-free blocking reagent or 10% goat serum in PBS 0.1% TritonX-100 (with some exceptions, see below), followed by incubation in primary antibody for 2 h at room temperature or overnight at 4 °C. Fluorescent Alexafluor-conjugated secondary antibodies were incubated for 1 h at room temperature. Sections were mounted in Prolong Diamond antifade with 4′,6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: Chicken anti-EGFP, 1:1000 (Abcam ab13970); mouse anti-Tuj1, 1:750 (Sigma-Aldrich T5076); goat anti-Sox10, 1:100 (Santa Cruz sc-17342); goat anti-Sox10, 1:100 (R&D Systems AF2864); mouse anti-Isl1, 1:50 (DSHB 40.3A4); rabbit anti-cleaved Caspase-3, 1:500 (Cell Signaling Technologies 9661); rabbit anti-FABP7, 1:200 (Cell Signaling Technologies 13347); goat anti-TrkA, 1:50 (R&D Systems AF1056); goat anti-TrkB, 1:200 (R&D Systems AF1494); goat anti-TrkC, 1:200 (R&D Systems AF1404); mouse anti-AP2α, 1:20, goat serum block (DSHB 3B5); sheep anti-DLL1, 1:200 (R&D Systems AF5026); rabbit anti-p75, 1:250 (Abcam 52987); goat anti-Jag1, 1:200 (R&D Systems AF599); rabbit anti-N1ICD, 1:100 (Cell Signaling Technologies 4147); sheep anti-Notch1, 1:200 (R&D Systems AF5267).

To examine cell proliferation, EdU (5-ethynyl-2′-deoxyuridine) (Life technologies) was prepared in 0.9% NaCl solution at 10 mg/ml. EdU was delivered by intraperitoneal injections to pregnant dams at E10.5 at 100 mg/kg of body weight and embryos were collected 1 h after injection.
Embryos were fixed in 4% formaldehyde overnight at 4 °C, cryopreserved in 20% sucrose, and embedded in OCT compound for cryosectioning. Sections were blocked in 10% Dako serum-free blocking reagent in PBS /0.1% TritonX-100, followed by incubation in primary antibody for 2 h at room temperature or overnight at 4 °C. Fluorescent Alexafluor-conjugated secondary antibodies were incubated for 1 h at room temperature. Sections were mounted in Prolong Diamond antifade with DAPI. The following primary antibodies were used: mouse anti-Tuj1, 1:750 (Sigma-Aldrich T5076); mouse anti-Isl1, 1:50 (DSHB 40.3A4); rabbit anti-cleaved Caspase-3, 1:500 (Cell Signaling Technology 9661); mouse anti-AP2α, 1:20 (DSHB 3B5); sheep anti-Dll1, 1:200 (R&D Systems AF5026); rabbit anti-N1ICD, 1:100 (Cell Signaling Technology 4147). Sections of EdU-labeled embryos were incubated with Click-iT EdU Kit Alexa-555 conjugated (Life Technologies) following staining with primary antibodies.