Anti cd31 antibody

Methods were followed as described (36 (link)). Formalin fixed paraffin-embedded sections of 2 mammary glands each from 5 mice were stained using the anti-CD31 antibody (Dianova). A 1:40 dilution of the antibody was used to stain the sections overnight. Diluted biotinylated anti-rat IgG (Vectastain kit) was added to the sections and incubated for 30 min. Vectastain ABC reagent (Vector) and 3, 30-diaminobenzamidine (DAB) was then used for color development. The vessel number, cumulative circumferential vessel length and cumulative vessel area were quantified using MetaVue software. For more details, refer to Supplementary Methods.

Xenograft tumors were collected on the day after the last dosing; tumor fragments were fixed with 10% formalin and embedded in paraffin, except for LI0334 tumor fragments, which were frozen in OCT compound and the tissue sections were fixed with cold acetone. Staining for endothelial cells with anti‐CD31 antibody and microvessel density (MVD) measurement were performed as described previously 25. Briefly, tissue sections were stained with anti‐CD31 antibody (Dianova, Hamburg, Germany) and the slides were scanned using the Aprio ScanScope XT system. Five regions of interest (ROIs, each 500 × 500 μm) with the highest densities of CD31‐stained microvessels were selected manually, and the number of microvessels in each ROI was measured.
Ki‐67‐positive cells were stained with anti‐Ki‐67 antibody (Cell Signaling Technology) and detected using Envision+ Single Reagents (Dako, Agilent Technologies, Santa Clara, CA). The percentage of Ki‐67 positive nuclei was quantified using HALO software (Indica Labs, Corrales, NM).

Recombinant human TGF-β1 LAP (rhLAP β1) and both monoclonal and polyclonal anti-human LAP β1 antibodies (MAB246 and AF246, respectively) were purchased from R&D Systems (Minneapolis, MN). Human PLK was from CalBiochem (San Diego, CA). Anti-αSMA antibody were purchased from DAKO (Glostrup, DK). Anti-CD31 antibody was from dianova GmbH (Hamburg, DE). Anti-F4/80 antibody was from AbD serotec, LLC (Oxford, UK). Anti-pSmad3C polyclonal antibody was a kind gift from Dr. Matsuzaki (Kansai Med. Univ., Japan). GST-rhLTGF-β1 protein was prepared as described follows. A plasmid encoding GST-rhLTGF-β1 was constructed by inserting a human TGF-β1 cDNA into the pGEX-6P-1 vector (GE Healthcare, Buckinghamshire, UK), and protein expressed in E. coli. BL21 (Stratagene, La Jolla, CA) and purified using Glutathione Sepharose (GE Healthcare).

Sections (5 μm) from full thickness skin pieces collected in four zones of the sample dedicated to histological analysis were stained with hematoxylin & eosin and Masson’s Trichrome. Immunostaining of blood vessels was performed with anti-CD31 antibody (#DIA310, Dianova GmbH, Hamburg, Germany) and revealed with a rabbit anti-rat biotinylated conjugated secondary antibody (#E0468) and streptavidin/horse radish peroxidase (#P0397) both purchased from Dako (Golstrup, Denmark).
The thickness of the dermis, hypodermis, and panniculus carnosus was measured on four hematoxylin & eosin-stained sections from each mouse at eight different locations of the section by image analysis using ImageJ software (NIH, Bethesda, MD, USA). The area occupied by CD31-positive vessels was measured in the three cutaneous layers (dermis, hypodermis, and panniculus carnosus) using ImageJ and expressed in percentages of the total field surface. The number of growing hair follicles^{11 (link)} was counted in four different skin sections of each mouse and expressed per unit length of epidermis.