S1700

Histological analysis was performed on the inferior lobe of the right lung. After fixation in 4% paraformaldehyde for 24 hours, the tissues were dehydrated in a series of graded ethanol solutions, then embedded in paraffin. Serial paraffin sections were cut in 4 μm thick and prepared for hematoxylin and eosin staining (for evaluation of whole tissue structure) or immunostaining (for inflammatory cells).
Before immunostaining, antigen‐retrieval was performed using antigen retrieval solution (S1700; Dako, Tokyo, Japan) and sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity. The sections were then incubated with Blocking One (Nacalai Tesque, Tokyo, Japan) for 30 minutes at room temperature to block unspecific binding of the antibodies. Primary antibodies against CD3 (1:300 dilution; rabbit monoclonal, Nichirei Corporation, #413601; Tokyo, Japan), and Myeloperoxidase (MPO, 1:300 dilution; rabbit polyclonal, Abcam, ab9535; Cambridge, UK) were detected with peroxidase conjugated appropriate secondary antibodies and a diaminobenzidine substrate. Images of the lungs were captured using a digital microscope camera (Keyence BZ‐X700) with ×4 and ×20 objective lenses.

Mice harboring orthotopic TE8-luc tumors were treated and euthanized 34 days after the treatment as scheduled in Figure 3A. 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.

Four-micrometer sections of formalin-fixed and paraffin-embedded specimens were deparaffinated, followed by antigen retrieval using Target Retrieval (S1700, Dako). The sections were subsequently blocked with phosphate-buffered saline plus 10% donkey serum and 3% bovine serum albumin and stained with anti–ACTA2-Cy3(1:400; C6198, Sigma-Aldrich), anti-collagen 1 alpha 1 (COL1A1) (1:200; ab279711, Abcam), anti-fibronectin 1 (FN1) (1:200; SAB5700724, Sigma-Aldrich), anti-pSMAD3 (0.5 μg/ml; LS-B64-50, LifeSpan BioSciences), anti-TAZ (1:400; ZRB1260, Sigma-Aldrich), anti-YAP (1:200; 4912, Cell Signaling Technology), anti-Ki67 (1:200; 14-5698-82, eBioscience), or anti-γH2AX (1:200; MAB15111, Abnova).

Mice were sacrificed on day 14, and tumors were fixed in 10% formaldehyde neutral buffer solution (Sigma-Aldrich, St. Louis, MO, USA) for 72 h and embedded in paraffin. Sections were rehydrated through an alcohol gradient and subjected to heat-mediated antigen retrieval using target retrieval solution S1700 (Dako, Santa Clara, CA, USA). The sections were stained with an anti-mouse CD8 antibody (Abcam, Cambridge, UK) followed by 3,3′-diaminobenzidine as the chromogenic substrate. For semiquantitative analysis, positive cells were counted in five fields/section.