Ab14917

For immunohistochemical analysis, paraffin-embedded pancreas sections (except those for LYVE-1) were processed for antigen retrieval in sodium citrate buffer (pH 6.0). Slides were incubated for 30 min with primary antibody against PCK-26 (1:500; Abcam, ab6401), CD-31 (1:50; Abcam, ab28364), LYVE-1 (1:50; Abcam; ab14917) or collagen-I (1:100; NB600-408, Novus Biologicals). After incubation with species-specific biotinylated secondary antibodies (1:200) for 30 min and streptavidin-HRP for 30 min (Vectastain, ABC kit; Vector Labs), the immunoperoxidase reaction was visualized by using the VECTOR NovaRED Peroxidase HRP Substrate Kit (Vector Labs). Slides were counterstained with hematoxylin QS and visualized with an Olympus IX70 inverted microscope and QImaging QCapture Pro 6.0 Software.

Mice were euthanized by CO₂ asphyxiation and necropsies. Skin
was removed as previously described or complete necropsies were performed (Silva, 2012 ). All tissues were fixed in
Fekete’s acid alcohol formalin solution for 12 hours and then processed
routinely. Tail was removed and oriented in longitudinal and cross sections
after decalcification. Tissues were serially sectioned and stained with either
hematoxylin and eosin (H&E), Masson’s trichrome, Verhoeff-Van
Geison, or Sirius red. H&E and Sirius red stained sections were reviewed
using both white and polarized light microscopy. Antibodies used for
immunohistochemistry included CD31 (for endothelial cells; Abcam, Cambridge MA,
cat# ab28364, 1:100), LYVE1 (for lymphatics, Abcam, Cambridge MA, cat#
ab14917, 1:100), and SMA (for vascular smooth muscle: 1:200, NB600-531, Novus,
Littleton, CO, USA or 1:400, A 2547, Sigma). Details are available on http://tumor.informatics.jax.org/html/antibodies.html. The
immunohistochemistry was done using a Ventana autostainer (Tuscon, AZ).
Diaminobenzidine (Sigma, St. Louis, MO) was used as chromogen.