Mouse anti n cadherin 3b9

Immunostaining was performed with modifications from standard protocol previously described (Nielsen and Dymecki, 2010 (link)). Primary antibody dilutions: rat anti-CD13 (MCA2183GA) (1:500) (AbD Serotec); mouse anti-desmin (D33) (1:50) (DAKO/Agilent); mouse anti-N-cadherin (3B9) (1:300) (Invitrogen); rat anti-Pdgfrβ (CD140b) (1:50) (Invitrogen). Secondary antibodies: Cy3 donkey anti-rat; Alexa647 donkey anti-mouse; Alexa647 donkey anti-rat (all dilutions 1:500) (Jackson ImmunoResearch). For EdU incorporation assay, mice were injected intraperitoneally with 10 μg/gram of body weight with EdU at P5–7 or P8–10 or P12–14. On the day of harvest (P7 or P10 or P14), tissue was harvested 2 h post-injection. Detection of EdU used Click-iT™ Plus chemistry, with AlexaFluor^® 647 component following manufacturer’s instructions (Invitrogen). Cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Tissue sections were mounted with ProLong Gold™ (Invitrogen) and coverslipped for imaging.

Protein lysates were prepared in 1X RIPA buffer from isolated cells (see above). Protein concentration was estimated using Precision Red (Cytoskeleton, Inc.) and NanoDrop One spectrophotometer. Proteins were separated using an 8% polyacrylamide separating/resolving gel, 4% stacking gel, and electrophoresis. Protein was transferred to PVDF membrane, and membrane was blocked with 3% bovine serum albumin in tris-buffered saline with Tween-20, before incubating with primary antibodies. Primary antibody dilutions: rabbit anti-gapdh (1:1,000) (Cell Signaling Technologies); mouse anti-N-cadherin (3B9) (1:1,000) (Invitrogen); rat anti-Pdgfrβ (CD140b) (1:1,000) (Invitrogen); rabbit anti-Rbpj (1:1,000) (Cell Signaling Technologies). HRP-conjugated secondary antibody dilutions: anti-mouse (1:1,000); anti-rabbit (1:1,000); anti-rat (1:1,000) (Cell Signaling Technologies). Chemiluminescent substate was applied to the membrane and bands were detected and quantified with BioRad ChemiDoc XRS+ and ImageLab software.