VEGF A - AB-90040

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WT and NG2KO mice at postnatal day 6 (P6), and adult naïve and EAE-affected WT and NG2KO mice at 20 and 40 dpi, were transcardially perfused with 100–150 ml of 2% paraformaldehyde (PFA) and 0.2% glutaraldehyde PBS solution, under deep anaesthesia (ketamine/xylazine cocktail, 90 mg and 4.5 mg/kg, respectively) by intraperitoneal injection. Whole brains were removed and post-fixed by immersion in the same fixative at 4°C for 4 h, washed in PBS overnight at 4°C, and stored in 0.02% PFA in PBS at 4°C. Using a vibrating microtome (Leica Microsystem), serial sagittal sections (30/35-μm thick) evenly spaced at 200 μm intervals, were cut from each hemisphere to allow the analysis of the entire antero-posterior extension of the cerebral cortex. The sections, in a range of 60 to 120 sections/hemisphere, were stored in a multiwell archive as free-floating sections in PBS at 4°C. The archived sections were submitted to immunostaining to ascertain the presence of demyelinating lesions, and the adjacent sections were then utilized for confocal morphometry (see for details [6]). Briefly, after permeabilization (0.5% Triton X-100 in PBS), free-floating sections were incubated with single or combined primary antibodies, overnight at 4°C, and with appropriate secondary antibodies (Table 1), for 45 min at RT, and then counterstained with TO-PRO-3 diluted 1:10k in PBS (Invitrogen). Finally, the sections were collected on Vectabond treated slides (Vector) and coverslipped with Vectashield (Vector). Negative controls were prepared by omitting the primary antibodies and mismatching the secondary antibodies. Sections were examined under Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems) using a sequential scan procedure. Confocal images were taken at 0.35 μm intervals through the z-, x-, and y-axes of the section, with 40x and 63x oil lenses.

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