Rabbit anti ng2 chondroitin sulfate proteoglycan

Pericytes and capillaries were stained in the granular layers of the cerebellar vermis and hemisphere slices as previously described [21 (link),42 (link)], focusing, respectively, on lobule V and lobule VI (see Supplementary Materials for details). The rabbit anti-NG2 chondroitin sulfate proteoglycan (Millipore, Merck KGaA, Darmstadt, Germany) and FITC-isolectin B4 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) primary antibodies were used to stain the pericytes and blood vessels, respectively. Slices were mounted on microscope slides, using ProLong^® Gold antifade reagent with DAPI (Molecular Probes, Thermo Fisher Scientifics, Waltham, MA, USA). Fluorescence of samples was observed with a TCS SP5 II LEICA confocal microscopy system (Leica Microsystems GmbH, Wetzlar, Germany) furnished with a LEICA DM IRBE inverted microscope. All acquisition files were visualized by LAS AF Lite software. Negative controls were carried out in parallel by treating slices with non-immune serum during the incubation procedures.

Cells, cultured on glass coverslips, were fixed in fresh 4% paraformaldehyde in PBS (0.1 M sodium phosphate buffer, 0.9% NaCl, pH 7.4) and subsequently blocked with 10% goat serum in PBS for 30 min, then incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. The following primary antibodies and dilutions were used: mouse anti-alpha-SMA, 1:200 (Sigma-Aldrich, #A2547); rabbit anti-CD31, 1:100 (abcam, Cambridge UK, #ab28364); rabbit anti-PDGFR-β 1:100 (abcam, #ab32570); rabbit anti-vWF, 1:200 (Santa Cruz Biotechnology, Dallas TX, #SC-365712); rabbit anti-calponin-1 1:100 (Millipore, Burlington MA, #04-589); rabbit anti-NG2 chondroitin sulfate proteoglycan, 1:100 (Millipore, #AB5320); rat anti-CD90, 1:200 (abcam, #ab3105), and mouse anti-3G5 McAb, 1:200 (prepared as described above from hybridoma cells, ATCC, #CRL-1814, stock concentration 2.96 µg/mL). Cells were washed with PBS + 0.1% Tween 20 and secondary antibody (Alexa 568-conjugated donkey anti-rabbit or IgG-Alexa 546-conjugated donkey anti-mouse (Life Technologies, Carlsbad CA, #A10042 and #A10036)) was applied in blocking buffer for 2 h at RT. Cell nuclei were labeled with Hoechst 33342 (Life Technologies). The coverslips were washed and mounted using ProLong Gold antifade reagent (Life Technologies, # P36935). Images were acquired with a confocal microscope (Nikon Eclipse Tie-A1RSi).