Immunohistochemical Analysis of Nestin-GFP/NG2-DsRed Mice

Frozen brains from Nestin-GFP/NG2-DsRed mice with or without injury were sectioned transversely into serial coronal sections 20 μm thick using a cryostat (Microm HM 500; Zeiss, Oberkochen, Germany) at -20°C, mounted on SuperFrost Plus Microscope Slides (Fisher Scientific) in series of six, and stored at -20°C before processing for immunocytochemistry. Sections were dried at room temperature for 1 hour, rehydrated in PBS, permeabilized with 0.5% Triton X-100 (Sigma) in PBS solution, and blocked to saturate nonspecific antigen sites using 5% (v/v) goat serum/PBS (Jackson Immunoresearch Labs) at 4°C overnight. The next day, the sections were incubated with anti-PDGFRβ antibody (gift from Dr W Stallcup, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA) at 1:100 dilution at room temperature for 4 hours and visualized using appropriate species-specific secondary antibodies. Hoechst 33342 was used to mark nuclei. The sections were mounted on slides using Fluorescent Mounting Medium (DakoCytomation) and examined with fluorescence microscopy [51 (link)].