Ab32570

PDGFR-β expression levels in the mouse brain tissue were detected with immunohistochemistry. Paraformaldehyde-fixed, paraffin-embedded specimens were removed, autoclaved, heat-treated in citrate saline buffer to extract antigens, and incubated with rabbit antibody (anti-PDGFR-β, 1:200, ab32570, Abcam) overnight at 4°C. All steps were performed according to the manufacturer’s protocols. Positive staining results were brown. Images of the immunohistochemically stained sections were analyzed with Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, United States).
PDGFR-β expression levels in the mouse brain tissue were examined with immunofluorescence staining. Paraffin sections were dewaxed to water, antigen-repaired, and serum-blocked. Then, the blocking solution was gently shaken off, primary antibody (anti-PDGFR-β, 1:200, ab32570, Abcam) was added dropwise to the sections, and the sections were incubated flat in a wet box overnight at 4°C. Then, the samples were incubated with secondary antibody before the nuclei were restained with DAPI. Next, the autofluorescence of the tissue was quenched, the sections were sealed, and the images were obtained under a fluorescent microscope. The DAPI-stained nuclei were blue under UV excitation and positively expressed as luciferin-labeled green fluorescence.

Formalin‐fixed/paraffin‐embedded samples from the patients included in this study were collected from Peking University People's Hospital. All the samples were cut into sections of 4‐μm thickness. The slides were deparaffinized in xylene for 30 min and rehydrated in absolute ethyl alcohol for 5 min (twice), 95% ethyl alcohol for 5 min, 75% ethyl alcohol for 2 min sequentially. Washed the slides with distilled water three times. A microwave‐oven is used for heat‐induced epitope retrieval, and during epitope retrieval the slides were I immersed in boiling EDTA buffer (pH 9.0; ZLI‐9069; Zsbio, Beijing, China) for 15 min. Antibody Diluent/Block (72424205; Perkin‐Elmer, MA, USA) was used for blocking. The antibodies used were anti‐CD4 (abcam, ab133616), anti‐CD8 (abcam, ab237709), anti‐CD19 (abcam, ab134114), anti‐CD57 (abcam, ab220187), anti‐CD11b (abcam, ab52478), anti‐CD31 (abcam, ab76533), anti‐pan cytokeratin antibody (abcam, ab32570) and anti‐PDGFR alpha + PDGFR beta (abcam, ab32570). The antigenic binding sites were visualized using the Opal 7‐Color Manual IHC Kit (Perkin‐Elmer, NEL811001KT) according to the manufacturer's protocol. Multicolour immunohistochemistry data were collected using a Mantra Quantitative Pathology Workstation (Perkin‐Elmer, CLS140089) and analysed by InForm (version 2.2.1).