Tissuefaxs 200

Mice were sacrificed at 6 hours, day 1, day 2, day 3, and day 7 after the virus challenge for collection of lung tissues. Lung tissues were fixed with 4% paraformaldehyde and then embedded in paraffin. Five-micrometer sections were cut and stained with H&E. The pathology scores were calculated following a methodology we previously published (He et al., 2020 (link)). For immunohistochemical (IHC) assays, paraffin sections of the lung were de-waxed and then subjected to heat treatment in citrate buffer, followed by quenching of endogenous peroxidase activity using 0.3% H2O2 in methanol. Sections were blocked for 1 hour (hr) with Fc Receptor Blocker and then incubated overnight at 4°C with F4/80 antibody (CST No.70076, 1:500). Antibody binding was detected using ZSGB System reagents (ZSGB, Beijing). After counterstaining with haematoxylin, the slides were incubated with CD4-specific antibody (Abcam No. ab183685, 1:1000) and CD8-specific antibody (LSBio No.LS-C43572, 1:200), and multiplexed immunofluorescence staining was then performed using Opal 7-color Manual IHC Kit (Akoya,USA). The stained slides were scanned by TissueFAXS 200 (TissueGnostics). The acquired images were analyzed by Strata Quest software to assess the number of F4/80+, CD4+, CD8+ cell and the inflammatory infiltration areas.

Mice were sacrificed on day 15 after tumor inoculation. The entire tumors were fixed with 4% paraformaldehyde and then embedded in paraffin. Sections of 5 µm in size were cut from the embedding tissue. The sections were further deparaffinized and rehydrated through xylene and graded ethanol series, and subjected to heat treatment in citrate buffer, followed by quenching of endogenous peroxidase activity using 0.3% H₂O₂ in methanol. Sections were blocked for 1 h with Fc Receptor Blocker and incubated separately with CD206 antibody (ab300621, 1:1000; Abcam) and F4/80 antibody (70076, 1:500; Cell Signaling Technology). Multiplexed immunofluorescence staining was then performed using Opal 7-color Manual IHC Kit (Akoya, USA). After washing, the stained slides were stained with DAPI for 2 min and scanned by TissueFAXS 200 (TissueGnostics). The acquired images were analyzed by Strata Quest software to assess the number of F4/80⁺ and CD206⁺.

MCF-10A cells were cultured in 3-D using Matrigel, as previously described [11 (link), 44 (link)]. Cell cultures in chamber slides were fixed with 4% paraformaldehyde, followed by staining for immunofluorescence using anti-ezrin antibody (3C12) from Santa Cruz Biotechnology (Santa Cruz, CA), Alexa 488-conjugated anti-mouse IgG and Hoechst 33342 from Life Technologies, Grand Island, NY.
For immunofluorescence using antibodies against the EMT markers and Ki67, MCF10A cells were densely plated on poly-lysine coated coverslips. After 14-day culture in minimal medium, cells were fixed and incubated with relevant primary antibodies and secondary fluorescein-conjugated horse anti-mouse IgG antibody (#FI-2000, Vector Laboratories, Burlingame, CA) or Texas Red^®-conjugated goat anti-rabbit IgG antibody (#TI-1000, Vector Laboratories). The slides were mounted with Vectashield Mounting Medium plus DAPI (#H-1200, Vector Labs)
Samples were then photographed using Nikon C2 Confocal Microscope or Zeiss Axiovert 200M Microscope. For quantification of the size of acini or “islands”, the images in10x magnification were acquired by TissueFAXS 200 (Tissuegnostics, Vienna, Austria) or by Zeiss Axiovert 200M, followed by analysis using the ImageJ software.