Vslide scanning microscope

Procedures for double staining with PDGFR‐β/α‐SMA and CD34 antibodies have been described earlier together with details of the α‐SMA detection antibody 5, 14, 15. The antibody used for PDGFR‐β detection was 28E1 Rabbit mAb, 3169, Cell Signaling Technology. CD34 stains were performed with the antibody clone QBEnd 10, M7165 Mouse mAb, DAKO 5, 14, 15. Detection of primary antibodies followed standard procedures as described previously. Stained slides were subsequently scanned using a Vslide scanning microscope (Metasystems, Alltlussheim, Germany).
Specificity of the PDGF‐β‐receptor antibody has been extensively characterized in earlier studies, including test‐staining of sections of paraformaldehyde‐fixed paraffin‐embedded cultured cells of known PDGF receptor status 19.

Tissue microarrays (TMA) of human temporal cortex were deparaffinized, rehydrated, and treated with antigen retrieval solution (pH 6.0) and peroxidase block solution in an automated Leica Bond RX immunostainer (Leica Biosystems). The TMAs were thereafter incubated with primary antibodies for 16 h at 4°C, washed, and blocked in Tris‐NaCl‐blocking buffer (TNB) before addition of secondary antibodies. Following this, tyramide signal amplification technology (TSA) cyanine 5 tyramide amplification reagent was added for 15 min before washing and quenching in 1% Sudan black. Fluorescence microscope images were acquired on a Vslide scanning microscope (MetaSystems, Alltlussheim, Germany) equipped with a CoolCube 2 camera, 2.5×, 5×, 10×, and 20× objectives and filter sets for DAPI (EX350/50–EM470/40), FITC (EX493/16–EM527/30), Cy3 (EX546/10–EM580/30), Cy3.5 (EX581/10–EM617/40), and Cy5 (EX630/20–647/long pass).
For a more thorough description of the methods including antibody validations such as sandwich immunoassays and immunocapture multiple sclerosis (MS), see Supporting Information.