Anti cd31 antibody

Tumors were cryosectioned or paraffin embedded. Paraffin-embedded tissue was H&E stained at the ULAM Pathology Cores for Animal Research at the University of Michigan and stroma quantified per low-power field (10x). Cryosectioned tumors were fixed and stained with anti-CD31 antibody (Dako) or permeablized and stained with anti-αSMA antibody- Cy3 (Abcam) and co-stained with mouse anti-human mitochondrial antibody (Life tech) with goat anti-mouse-alexa 488 secondary. CD31 + cells were counted to determine microvessel density per hpf (40x). Anti-αSMA staining was quantified by determining the area of positive staining per hpf (40x). 10 sections from 3 tumors per treatment group were analyzed [58 (link)].

This study was approved by the Bioethics Committees of Gifu University (28-149). Formalin-fixed paraffin-embedded surgically resected tissue specimens from RCC patients (n = 20) were immunohistochemically analyzed for PSMA and CD31 expression. After deparaffinization, the tissue sections (4 µm) were processed in the Ventana benchmark ultra automatic stainer (Ventana Medical Systems, AZ, USA) according to the manufacturer’s instruction. Briefly, the antigen retrieval was conducted in Cell conditioning 1 (CC1) buffer (Tris–EDTA, pH 8.5, Ventana) for 60 min at 95 °C. The sections were then incubated with anti-PSMA antibody (Abcam, 1:500 in dilution) or anti-CD31 antibody (Dako, CA, USA, 1:100 in dilution) for 1 h at 25 °C, followed by amplification and visualization using I-View DAB universal kit, which is based on the labeled streptavidin–biotin method (Roche, Basel, Switzerland). The sections were counterstained with Mayer’s hematoxylin. Negative control tissue sections were prepared by omitting the primary antibody. PSMA staining was scored by the intensity (1, 0+; 2, 1+; 3, 2+; 4, 3+) and percentage (1, 0–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%) under 200× magnification. The score of intensity multiplied by that of percentage was used as the score for PSMA expression.

Immunohistochemical analysis was performed on 4-μm-thick tissue sections cut from formalin-fixed paraffin-embedded surgical specimens. The sections were stained with hematoxylin and eosin (HE) or with anti-CD31 antibody (1:200 dilution; DAKO, Glostrup, Denmark) using a VENTANA BenchMark XT automated staining device (Ventana Medical System, Tucson, AZ, USA), according to the manufacturer’s instructions. The Vessel area was determined by CD31 positive staining normalized to the total area fraction. The number of microvessels was counted as described previously [62 (link)]. Briefly, the most vascularized areas were selected and counted in a ×400 field. Any single or cluster of endothelial cells that were separated clearly from adjacent microvessels was considered as one countable microvessel. The average counts from the three most vascularized areas were recorded in each case to measure the degree of angiogenesis.