Rat anti endomucin

Pancreas preparation and antibody labeling were as described (33 (link)). Antibodies used were as follows: hamster anti-CD11c (clone N418 1:300, eBioscience); rat anti-F4/80 (clone MCA4976 1:200, BioRad), rabbit anti-insulin (1:500, Cell Signaling); rat anti-endomucin (1:500, Santa Cruz Biotechnology); rabbit anti-fibronectin (clone AB1942 1:5,000, Chemicon); and mouse anti-collagen I (1:300, Abcam). Nuclei were labeled using dapi (Sigma). One to four slices were randomly selected from >3 animals/groups. Images were acquired using a Zeiss LSM 780 confocal microscope and analyzed using Imaris (Bitplane) and ImageJ (NIH).

The following primary antibodies were used for immunohistochemistry or immunofluorescence staining of tumors: goat anti-LYVE-1 (R&D Systems, cat no. AF2125), rat anti-endomucin (Santa Cruz, cat no. sc-65495), Cy3-conjugated mouse anti-smooth muscle actin (Sigma, cat no. C6198), and hamster anti-podoplanin (abcam, cat no. ab11936). All secondary antibodies were purchased from Jackson ImmunoResearch.

Pancreas preparation and antibody labeling were as described (33 (link)). Images were acquired using a Zeiss LSM 780 confocal microscope. Images were analyzed using Imaris (Bitplane), Volocity (Perkin Elmer) and ImageJ (NIH). For quantifications, four slices were randomly selected from at least three animals/group, and all islets present analyzed. A priori, this is sufficiently-powered to detect a minimum 1.2-fold difference with a SD of 40%, a power of 0.9, and alpha = 0.05 (G*Power 3.1). Antibodies used were: rabbit anti-Ki67 (1:200, CliniSciences), guinea-pig anti-insulin (1:400, Abcam), mouse anti-glucagon (1:200, Sigma), rat anti-endomucin (1:500, Santa Cruz Biotechnology), anti-PDGFRβ (1:200, R&D Systems); alexa 647-conjugated anti-αSMA (1:200, Abcam). Nuclei were labeled using dapi (Sigma). To measure islet pericyte core area, single plane confocal images were used, and the surface occupied by a DsRed pericyte cellular body containing a nucleus was measured using ImageJ. Number of pericyte cellular bodies containing a nucleus on single plane images were counted and divided by islet surface or exocrine tissue surface in the image. The proportion of proliferative beta cells was obtained by dividing number of Ki67+ nuclei by total number of nuclei of insulin+ cells in islets, as described (33 (link)).

The following primary antibodies were used for immunoblotting or immunofluorescence staining: goat anti-Lyve1 (R&D Systems, #AF2125; dilution 1:250), rabbit anti-phospho AKT (Ser 473, Cell Signaling, 4060S; dilution 1:1000), rabbit anti-AKT (Cell Signaling, 4685; dilution 1:1000), rabbit anti-phospho ERK1/2 (Cell Signaling, 4370; dilution 1:1000), rabbit anti-ERK1/2 (Cell Signaling, 4695; dilution 1:1000), rabbit anti-tubulin (Cell Signaling, 2148; dilution 1:2000), rabbit anti-GAPDH (Cell Signaling, 2118; dilution 1:1000), chicken anti-GFP (abcam ab13970; 1:4000), rat anti-endomucin (Santa Cruz, sc-65495; dilution 1:50), rabbit anti-Prox1 (AngioBio, 11-002; dilution 1:1000), rabbit anti-neuropilin-2 (Cell Signaling, 3366; dilution 1:500), rabbit anti-Runx1 (Abcam, ab92336; dilution 1:1000), and rabbit anti-KRAS^G12D (Cell Signaling, #14429; dilution 1:1000).