Rabbit anti pdgfr β

Brain slices prepared from the indicated groups of mice were subjected to double immunofluorescence staining. The primary antibodies used in our studies were mouse anti-β-amyloid (20.1) (1:25, Santa Cruz Biotechnology), rabbit anti-PDGFRβ (1:100, Abcam), and rabbit anti-CD36 (1:100 ABclonal). All images were obtained, and colocalization was visualized, using a Nikon confocal microscope.

Cells were lysed in a protein extraction solution (Intron, Seongnam, South Korea). After centrifugation, the samples were boiled at 95 °C for 10 min, and 50 µg protein of each lysate was subjected to electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels. All samples were electroblotted on polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA) and, after blocking, the blots were incubated with appropriate rabbit anti-THBS4 (Santa Cruz Biotechnology, sc-390734), rabbit anti-PDGFRβ (Abcam, Cambridge, MA, USA, ab32570), mouse anti-phosphorylated PDGFRβ (Santa Cruz Biotechnology, sc-365464), rabbit anti-PDGF-D (Abcam, Cambridge, MA, USA, ab234666), mouse anti-IP3R (Santa Cruz Biotechnology, sc-271197), and rabbit anti-STIM1 (Cell Signaling Technology, Danvers, MA, USA, 5668S) antibodies in TBS-T (TBS-0.05% Tween 20) for 90 min, which was followed by washing three times with TBS-T for 15 min each, and incubation with horseradish peroxidase-conjugated anti-mouse or rabbit immunoglobulin G antibodies for 1 h. After further washing, the blots were incubated for 3 min with Western blotting HRP-substrate (Merck Millipore), and chemiluminescence was detected after exposure of the filters to ECL-Western blot films for 10 s to 10 min. Original Western blots are shown on Figures S7–S14.

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).