Buffered formalin solution

At euthanization, kidneys were collected, cut sagittally into halves, and fixed in 10% buffered formalin solution (Sigma, St. Louis, MO, USA) for 48 h. Following fixation, kidney halves were washed in 100% ethanol and embedded in paraffin. Kidney halves were cut into 5–7 μm sections, which were deparaffinized, rehydrated, and permeabilized with 0.1% Triton solution (BioRad, Hercules, CA, USA). Tissue was blocked with 10% AquaBlock solution (EastCoastBio, North Berwick, ME, USA) before being immunolabeled with the lymphatic endothelial cell markers LYVE-1 or podoplanin (goat polyclonal; R&D Systems, Minneapolis, MN, USA) by overnight incubation at 4 °C. Alexafluor 488 or 594 (Life Technologies, Carlsbad, CA, USA) secondary antibodies were used to visualize lymphatic vessels. Samples were incubated with appropriately conjugated secondary antibodies for an hour at room temperature. Negative controls were incubated with only a secondary antibody. All labeled slides were mounted with ProLong Gold anti-fade reagent containing DAPI (Invitrogen, Carlsbad, CA, USA). An Olympus BX51 fluorescence microscopy system with Olympus Q5 camera was used for imaging. Images were captured at 40× magnification using Olympus CellSens imaging software (Olympus, Shinjuku, Tokyo, Japan). The same methods were utilized to image lymphatics in the heart, liver, lung, and skin (10× or 20× magnification).

All animals were weighed and examined externally. The cranial, thoracic, and abdominal cavities were opened for macroscopy. The brain, heart, liver, thymus, kidneys, adrenals, spleen, testes or ovaries of dogs and rats were weighed at the end of the experiment. In addition, the dog prostate was weighed. Samples of organs and tissues were collected from each animal into a 10% buffered formalin solution (Sigma, USA). Five micrometer thick slices were prepared from the fixed samples, stained with hematoxylin-eosin, and examined under a microscope (AxioScopeA1, Carl Zeiss, Germany).

Collected tumor tissues from dogs and xenograft were fixed in 10% buffered formalin solution (Sigma-Aldrich, St Louis, MI) and paraffin embedded for routine pathological analysis. Immunohistochemical staining was carried out on 4- to 6-μm-thick slides using an automated protocol developed for the Discovery XT automated slide staining system (Ventana Medical Systems, Inc.). Alongside haematoxylin–eosin staining (HE), the following commercially available antibodies were used for the phenotypical characterization of melanocytes: Melan-A (Thermo Fisher Scientific, Waltham, MA), S-100 protein (Dako, Agilent Technologies, Denmark), Vimentin, and cytokeratin (Biogenex Laboratories, San Ramon, CA) (see Additional file 1). Tumor sections were deparaffinized and incubated for 1 h with the appropriate antibody before incubation with Discovery UltraMap anti-Rabbit (760–4315, Roche, Basel, Switzerland) or anti-mouse horseradish peroxidase (HRP) (760–4313, Roche, Basel, Switzerland) secondary antibodies and the Discovery ChromoMap DAB kit reagents (760–159, Roche, Basel, Switzerland) (Additional file 1).

Mouse tumor tissues were fixed in 10% buffered formalin solution (Sigma-Aldrich) at room temperature and infiltrated with 20% sucrose overnight at 4 °C. Tumor tissue blocks were then imbedded with OCT Tissue-Tek compound (SAKURA, West Chester, PA, USA) onto a PVC plastic mold and rapidly frozen to −30 °C in dry ice, 15 μm thick sections (at least 10 slices per block) were obtained using a Leica CM1850 cryostat (Leica Microsystems, Wetzlar, Germany). Frozen sections were dried for 2 h at room temperature, washed with PBS three times for 3 min, and surface tensions reduced using an application of 0.05% triton X-100 2 times for 5 min. Sections were blocked in 5% normal goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h, incubated overnight in a humidified chamber with each primary antibody, 1:200 Rat-anti mouse PECAM (CD31) and Rat-anti mouse VE-Cad (CD144) (Both from BD bioscience, La Jolla, CA, USA), washed with PBS with 0.1% Triton X-100, and then incubated for 1 h with secondary antibodies 1:500 FITC goat anti-rat IgG (BD bioscience) at room temperature. After washing with PBS, samples were mounted with ProLong Gold anti-fade regent containing 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen), and confocal fluorescence images were acquired using the FV10i FLUOVIEW Confocal Microscope (Olympus, Tokyo, Japan).