Peroxidase conjugated secondary antibodies

VR-EPCs were homogenized in lysis buffer (Cell Signaling, Massachusetts, USA) containing a cocktail of protease inhibitors, and the protein concentration was determined by Lowry assay (BioRad, Germany). Samples were separated in 10% PAGE-SDS gels. After transfer to nitrocellulose membranes, samples were blocked in 5% albumin and incubated with primary antibodies for 2 hours at room temperature. After 4 washing steps with PBS, membranes were incubated with peroxidase-conjugated secondary antibodies (Dianova, Hamburg, Germany). Finally, detection was performed using a chemiluminescence system (GE Healthcare, Germany).

Anesthetized mice were transcardially perfused using PBS followed by 4% paraformaldehyde (PFA). Brains were extracted and post-fixed in 4% PFA overnight. Free-floating 50 µm vibratome (Leica, Wetzlar, Germany) sections were incubated overnight with anti-IBA1 (1:1000, 019-19741, Wako, Japan). Alexa Flour 488-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) were used at 1:200 for 2 h at room temperature. Nuclei were counterstained using 4′6-diamidino-2-phenylindole (DAPI, Roche, Basel, Switzerland) and sections were mounted on glass cover slips. Imaging was performed using the Leica TCS SP8 confocal laser scanning microscope (Leica, Wetzlar, Germany) and LAS AF image analysis software (Leica, Wetzlar, Germany). For DAB (3,3′-diaminobenzidine) staining, peroxidase-conjugated secondary antibodies (Dianova, Hamburg, Germany) were used. Images were captured using A Zeiss AxioImager I (Zeiss, Göttingen, Germany) and the Stereoinvestigator Software 8.0 (MicroBrightField, Magdeburg, Germany).