Immunohistochemistry Protocol for Tissue Analysis

Immunohistochemistry was performed on 50 μm tissue sections as previously described (Moldrich et al., 2010 (link)). Primary antibodies used: sheep anti-DRAXIN (1:250; AF6149, R&D Systems), mouse anti-human KI67 (1:500; 550609, BD Pharmingen), mouse anti-GAP43 (1:500; MAB347, Millipore), rabbit anti-GFAP (1:500; Z0334, Dako), mouse anti-GLAST (or EAAT1; 1:500; ab49643, Abcam), rabbit anti-GLAST (or EAAT1; 1:250; ab416, Abcam), chicken anti-LAMININ (1:250; LS-C96142, LSBio), rabbit anti-LAMININ (1:250; L9393, Sigma), rat anti-NESTIN (AB 2235915, DSHB), and rabbit anti-SOX9 (1:500, AB553, Merck). Secondary antibodies were Alexa Fluor IgG antibodies (1:500, Invitrogen) or biotinylated IgG antibodies (1:500 or 1:1000, Jackson Laboratories) used in conjunction with Alexa Fluor 647-conjugated streptavidin (1:500, Invitrogen) amplification. EdU labelling was performed using the Click-iT EdU Alexa Fluor 488 or Alexa Fluor 555 Imaging kits (Invitrogen) according to the manufacturer’s instructions. Cell nuclei were labelled using 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen) and coverslipped using ProLong Gold anti-fade reagent (Invitrogen) as mounting media.