Rabbit anti lyve 1

Formalin-fixed paraffin-embedded uveal tumor sections (10 μm) were immunolabeled with Rabbit anti-S100B (DAKO) and anti-CD31 (Biolegend) antibodies to identify melanoma cells and blood endothelial cells, respectively, as described previously [16 (link)]. For analysis of proliferating cells, blood and LVs in uveal tumors, cryosections were stained with rat anti-Ki67-biotinalyted (eBioscience), armenian hamster anti-CD31 (PECAM-1, Millipore) or rat anti-CD31 (PECAM-1, BD Biosciences) and rabbit anti-Lyve-1 (Abcam) antibodies. Cy3-conjugated streptavidin, Alexafluor647-conjugated or Cy3-conjugated anti-armenian hamster and Alexafluor488-conjugated anti-rabbit (Jackson ImmunoResearch Laboratories) antibodies were used for detection. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for cell nuclei visualization and mounted for analysis. Immunofluorescent labelled specimens were viewed with a fluorescence wide field (Axio Imager.Z1, Axioxam HRM camera; Carl Zeiss Micro Imaging, Jena, Germany). Tumor areas and BV size were measured using ImageJ software.

After DSS administration, the fluorescent membrane dye CM-Dil (5 μM in 2 μL of PBS per PP) (Life Technologies, USA) was injected into Peyer’s patches (PPs) surrounding the ileum [19 (link), 27 (link)]. After two-photon imaging, mice (n = 6 per group, three male and three female) were sacrificed and the meninges isolated for immunohistological analysis of CM-DiI labeling as described previously [28 (link)]. Briefly, mandibles and the skull rostral to the maxillae were removed, and the top of the skull with the meninges was collected and fixed in 4% paraformaldehyde (PFA) for 24 h at 4 °C. The meninges were dissected away from the skullcap, simultaneously permeabilized with 0.3% Triton X-100 and blocked with 10% goat serum for 1 h at room temperature, and then incubated overnight at 4 °C with primary rat anti-NLRP3 (1:100, Thermo Fisher, USA), rabbit anti-CD3 (1:100, Abcam, USA), and rabbit anti-LYVE-1 (1:100, Abcam, USA). Immunolabeled tissues were then incubated with an Alexa Fluor® 555-conjugated anti-rabbit IgG [(H+L), F(ab')2 Fragment (1:300, Cell Signaling Technology)] and Alexa Fluor® 488-conjugated anti-rat IgG [(H+L) (1:300, Cell Signaling Technology, USA)] in PBS containing 10% normal goat serum at room temperature for 1 h. Fluorescence images were acquired using a confocal microscope (Leica, Germany).

10 μm-thick cryopreserved tumors, skin and muscles sections (N=5 sections for each sample) were fixed with PFA 4% and incubated overnight at 4°C with the following primary antibodies: rat anti-mouse PECAM-1 (anti-CD31; 1:200; BD Pharmingen), rabbit anti-LYVE-1 (1:100, Abcam), rat-anti-LYVE-1 (1:100, Novus Biologicals), goat anti-mouse PlGF-2 (1μg/ml, R&D Systems). Isotype IgG for anti-LYVE1 was substituted for the primary antibody to assess the specificity of the staining. Bound antibody was detected with Alexa fluor-conjugated secondary antibodies. Sections were mounted with Vectashield with DAPI (Vector Laboratories). Images were recorded with a digital camera Leica.

PLNs were embedded in optimum cutting temperature compound and stored at −80 °C. Then, the frozen sections were cut at 8μm and washed with PBS for 5 min. Samples were blocked with 3% (vol/vol) bovine serum albumin in PBS and incubated with primary antibodies overnight. Sections were washed 3 times with PBS and incubated with secondary conjugated antibodies at room temperature. The following antibodies were used for the staining: goat anti-PDPN (R&D Systems, 1:200), rat anti-Meca79 (Novus Biologicals, 1:200), rabbit anti-Fibronectin (Abcam, 1:300), rat anti-ERTR7 (Santa Cruz Biotechnology, 1:100), rabbit anti-collagen IV (Abcam,1:300), rat anti-B220 (Invitrogen, 1:200), rabbit anti-CD3 (Abcam, 1:250), rabbit anti-lyve-1 (Abcam, 1:300). Secondary antibodies were either FITC- or Cy3-conjugated (Jackson ImmunoResearch, 1:200). DAPI (VECTASHILED, Vector Laboratories) mixed in Prolong-Gold mounting media was used as a nuclear counterstain. Images were obtained by EvosFL Auto2 microscopy. All images were automatically processed using ImageJ (NIH) and split into RGB channels. Auto threshold was used to convert intensity values of the immunofluorescent staining into numeric data.