Prb 160p 100

The primary antibodies used were: rabbit anti-TRPM8 (Ab109308, Abcam), mouse anti-p21 (Clone SX118, Dako), rabbit anti- cytokeratin 14 (PRB-155P-100, Covance), rabbit anti- cytokeratin 5 (PRB-160P-100, Covance), mouse anti-cytokeratin 18 (Sc-51582, Santa Cruz) and mouse anti-Vimentin (Clone V9, Dako).

Whole-mount inmunostaining was performed as described (Gaete and Tucker, 2013 (link)), with minor modifications: permeabilization and washes were performed using a PBS-Tr solution containing 1% Triton X-100. DNA was stained with DAPI 1:1000 and samples were mounted using Vectashield. The following antibodies and dilutions were used: Sox2 1:300 (2748s, Cell Signalling), Sox9 1:300 (AB 5535, Millipore), Keratin-5 1:500 (PRB-160P-100, Covance), E-cadherin 1:200 (10R-27500, Fitzgerald), Laminin 1:200 (L9393, Sigma). Alexa Fluor 561 and 488 1:500 were used as secondary antibodies. For BrdU assay, intraperitoneal injection of 10 mg/kg of BrdU was performed in a pregnant female carrying E13.5 embryos. The mother was culled and embryos fixed 24 h after injection. Double BrdU and Sox2 detection was performed as described (Gaete et al., 2012 (link)) with the same minor modifications described above for immunostaining.