The brains of E20 (for CD31, MMP2 and MMP9), P0 (for hematoxylin and eosin and collagen IV), or P52 (for Luxol Fast Blue, myelin basic protein and SMI-312) rats that had been perfused with 10% neutral buffered formalin were post-fixed 2–3 days in paraformaldehyde. Brains were transferred to 30% sucrose for cryopreservation then frozen in OCT. Cryosections (10 μm) were stained with hematoxylin and eosin (H&E) or Luxol fast blue (LFB) following standard protocols [28 (link)].
Immunohistochemistry was performed as described [20 (link),28 (link)]. Sections were incubated overnight with primary antibodies, including: goat anti-CD31 (PECAM-1) (1:200, sc-1506; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-MMP2 (1:100; sc-10736; Santa Cruz); rabbit anti-MMP9 (1:200; #ab38898; Abcam, Cambridge, MA); rabbit anti-collagen IV (1:400; #ab6586; Abcam); rat anti-myelin basic protein (MBP) (1:200; #ab7349; Abcam); mouse anti-SMI-312 (1:200; #837901; BioLegend, San Diego, CA) at 4°C. After several rinses in PBS, sections were incubated with species-appropriate fluorescent secondary antibodies (Alexa Fluor 488 and 555, Molecular Probes, Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Controls included the omission of primary antibodies.
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