Donkey anti rat rabbit goat

For frozen sections, OCT was removed by incubation with 1× PBS. We permeabilized cells in ice-cold MeOH for 10 min, followed by incubation in normal donkey serum (Jackson ImmunoResearch, Cat# 017-000-121; RRID:AB_2337258) and 0.3% Triton-X100 (Sigma-Aldrich, Cat# X100). Ki67 (1:200, Cell Signaling Technologies, Cat# 12202S; RRID:AB_2620142), anti-CNPAse (1:250, Sigma-Aldrich Millipore, Cat# AB9342; RRID:AB_569543); β-galactosidase polyclonal antibody (1:1000, Thermo Fisher, Cat#: A-11132; RRID:AB_221539). All secondary antibodies were donkey anti-Rat/Rabbit/Goat from Jackson ImmunoResearch, reconstituted in 50% glycerol and used at 1:250 dilution. To visualize nuclei, sections were stained with DAPI for 10 min, washed with PBS and mounted in FluoromountG (Electron Microscopy Sciences, Hatfield, PA). Images were acquired with ImageJ Acquisition software using a fluorescence microscope (Axiovert 200M) with 10×/0.4 or 40×/0.6 objectives (Carl Zeiss, Inc), or with NIS-Elements software using confocal microscopy (Nikon).

For frozen sections, OCT was removed by incubation with PBS. We permeabilized cells in ice cold MeOH for 10 min., followed by incubation in normal donkey serum (Jackson ImmunoResearch cat# 017–000-121) and 0.3% Triton-X100 (Sigma-Aldrich Cat# X100). Primary antibodies and dilutions were: Ki67 [11F6] (1:200, Biolegend cat# 151202), MBP (1:200 SCBT cat# sc-13,914), Krox20 (1:400 Abcam cat# ab43020), Sox10 (1:100 SCBT cat# sc-17,342), S100 (1:1000 Agilent Technologies Cat# Z031129–2), S100β [EP1576Y] (1:1000 Abcam cat# ab52642) All secondary antibodies were donkey anti Rat/Rabbit/Goat from Jackson ImmunoResearch, reconstituted in 50% glycerol and used at 1:250 dilution. To visualize nuclei, sections were stained with DAPI for 10 min., washed with PBS and mounted in FluoromountG (Electron Microscopy Sciences, Hatfield, PA). Images were acquired with ImageJ Acquisition software using a fluorescence microscope (Axiovert 200 M) with 10x/0.4 or 40x/0.6 objectives (Carl Zeiss, Inc.), or with NIS-Elements software using confocal microscopy (Nikon).