Cryojane taping system

Mice were anesthetized and the tissues harvested (liver, spleen, small intestine and omentum [tumor cells]) for immunofluorescence (IF) or immunohistochemistry (IHC) staining. Organ slices were cryo-preserved in 30% sucrose in PBS at 4 °C overnight for frozen tissues, embedded in NEG50 mounting medium (Thermo Fisher Scientific). The tissues were then frozen in a liquid nitrogen pre-chilled 2-methylbutane. Sectioning of frozen tissues was carried out using the CryoJane taping system (Leica Biosystems Inc). All tissues slides were subject to IHC analysis with specific primary Abs (Armenian hamster anti-CD31 [1:1000; catalog number MAB1398Z; MilliporeSigma], rat anti-endomucin [1:1000; catalog number 14-5851-81; eBioscience], human anti-B7-H3 (CD276) [1:200, catalog number AF1027; R&D Systems antibody], rabbit anti-firefly luciferase [1:200, catalog number ab21176; Abchem], and used the Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for images acquisition, in accordance with the standard protocols as published previously [7 (link),30 ]. Images were analyzed by CellSens Dimension imaging software (Olympus Soft Imaging Solutions).

Mice were administrated with 1×10 ¹¹ VP of triple targeted Ad via tail-vein injection. Three days post virus infection, mice were anesthetized and tissues (Liver, Lung, Pancreas, Spleen, Kidney, Small Bowel, Heart, Muscle and Brain) harvested for immunofluorescence staining. For frozen sections, organ slices were cryo-preserved in 30% sucrose in PBS at 4^oC overnight, embedded in NEG50 mounting medium (Thermo Fisher Scientific), and then frozen in a liquid nitrogen pre-chilled 2-methylbutane containing bucket. Sectioning of frozen organs was carried out using the CryoJane taping system (Leica Biosystems Inc). All frozen section slides were subject to immunofluorescence staining analysis in accordance with the standard techniques (8 (link)). Primary antibodies used in this study included hamster anti-CD31 (EMD Millipore), rat anti-endomucin (eBioscience), rat anti-PDGFRβ (eBioscience), rabbit anti-HSVtk (Dr. Summers’s lab) and Alexa Fluor 488 or 594-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).