Wha 800281

Dorsolateral skin from embryos were microdissected and placed on nucleopore filters (VWR, WHA 800281) and fixed overnight with 4%PFA at 4° C. Skin explants were blocked for 6 hours in 5% normal donkey serum, 1% bovine serum albumin and 0.5% Triton-X100 at RT. Explants were incubated overnight at 4° C in the following primary antibodies: chicken anti-RFP (1:250, NovusBio, NBP1–97371), rabbit anti-Sox2 (1:400, Abcam, ab97959), rabbit anti-Sox9 (1:500, Millipore, ab5535), goat anti-Pcadherin (1:500, R&D Systems, AF761), rabbit anti-RFP (1:500, Rockland, 600–401-379), Ki-67 Monoclonal Antibody (SolA15) (1:200, Invitrogen, 14–5698-82). Explants were then washed in 0.2% Tween20/PBS for 4–6 hours on a rotator and then incubated with the following secondary antibodies: Alexa Fluor 488-donkey anti-goat (1:200, Invitrogen, A11055), Alexa Fluor 568-donkey anti-rabbit (1:200, Invitrogen, A10042), Alexa Fluor 488-donkey anti-rabbit (1:200, Invitrogen, A21206), Alexa Fluor 647-goat anti-rat (1:200, Invitrogen, A-21247), Alexa Fluor 405-donkey anti-rat (1:200, Invitrogen, A-21208) overnight at RT. Washes were carried out for 6 hours with 0.2% Tween20/PBS. Hoechst (1:750 in PBS) was used for 45 min before mounting on slides with SlowFade Gold Antifade Mountant (Invitrogen, S36936).

Dorsolateral skin from embryos were microdissected and placed on nucleopore filters (VWR, WHA 800281) and fixed overnight with 4%PFA at 4° C. Skin explants were blocked for 8 hours in 5% normal donkey serum, 1% bovine serum albumin and 0.5% Triton-X100 at RT. Explants were incubated overnight at 4°C in the following p rimary antibodies: rabbit anti-Sox2 (1:200, Abcam ab97959), rabbit anti-Sox9 (1:500, Millipore, ab5535), rabbit anti-Cytokeratin17 (1:1000, Abcam ab53707), goat anti-Pcadherin (1:400, R&D Systems, AF761), rabbit anti-Ccna2 (1:500, Abcam ab181591), chicken anti-GFP (1:500, Abcam ab13970). Explants were then washed in 0.2% Tween20/PBS for 6 hours on a rotator and then incubated with the following secondary antibodies: Alexa Fluor 488-donkey anti-goat (1:200, Life technologies, A11055), Alexa Fluor 568-donkey anti-rabbit (1:200, Life technologies, A10042), Alexa Fluor 488-donkey anti-rabbit (1:200, Life technologies, A21206) overnight at RT. Washes were carried out for 6 hours with 0.2% Tween20/PBS and explants were mounted on slides with Vectashield DAPI. Explants were imaged in 3 dimensions using the LaVision TriM Scope II (LaVision Biotec) microscope.