Mice harboring orthotopic TE8-luc tumors were treated and euthanized 34 days after the treatment as scheduled in Figure 3 A. Orthotopic tumors were harvested, fixed in 10% formaldehyde neutral buffer solution (Sigma-Aldrich, St Louis, MOSA) for 72 h, and embedded in paraffin. Sections (5-μm thick) were rehydrated using an alcohol gradient and subjected to heat-mediated antigen retrieval using target retrieval solution S1700 (Dako, Santa Clara, CA).
Sections were mounted on silanized slides (Dako Cytomation, Glostrup, Denmark) and stained with HE. Sequential sections were subjected to immunohistochemical analysis to detect HSV-1. The sections were treated with peroxidase blocking solution (Dako) and Blocking One (Nacalai Tesque, Kyoto, Japan), incubated with a rabbit polyclonal anti-HSV-1 antibody (1:2,000 dilution, 3 μg/mL) (Dako Cytomation), rinsed, and incubated with an HRP-conjugated goat anti-rabbit IgG antibody (Nichirei Bioscience, Tokyo, Japan). The sections were developed with 3,3′-diaminobenzidine (DAB) Peroxidase Substrate kit (Vector laboratories, Burlingame, CA), and then counterstained with hematoxylin. A NanoZoomer Digital Pathology slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan) was used to view slides.
Sections were mounted on silanized slides (Dako Cytomation, Glostrup, Denmark) and stained with HE. Sequential sections were subjected to immunohistochemical analysis to detect HSV-1. The sections were treated with peroxidase blocking solution (Dako) and Blocking One (Nacalai Tesque, Kyoto, Japan), incubated with a rabbit polyclonal anti-HSV-1 antibody (1:2,000 dilution, 3 μg/mL) (Dako Cytomation), rinsed, and incubated with an HRP-conjugated goat anti-rabbit IgG antibody (Nichirei Bioscience, Tokyo, Japan). The sections were developed with 3,3′-diaminobenzidine (DAB) Peroxidase Substrate kit (Vector laboratories, Burlingame, CA), and then counterstained with hematoxylin. A NanoZoomer Digital Pathology slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan) was used to view slides.