Prolong gold and dapi

Mouse thyroids were quickly dissected and immersion-fixed in 10% formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (Vector Laboratories). For immunofluorescence, thyroid sections (6 μm) were deparaffinized in Citrisolv, then rehydrated using a graded ethanol series, followed by antigen retrieval in citrate buffer, blocking in 1.5% goat serum, incubation with primary antibodies (overnight, 4°C) and Alexa Fluor–conjugated secondary antibodies (Invitrogen A11073, A11001, A21422, A21428, A21245; Jackson ImmunoResearch 712-606-153; 1 hour, room temperature), counterstaining with ProLong Gold and DAPI (Invitrogen), and imaging with Nikon A1 confocal microscope or Leica STELLARIS 8 FALCON confocal microscope. Quantification of CD45⁺ cells in the proportion of total cells was performed using AIVIA Artificial Intelligence-guided Software. Immunohistochemistry of Ki67 used VECTASTAIN-ABC (Vector Laboratories) with 40× objective image capture (Olympus EX51 Microscope). Quantitation of Ki67-positive nuclei as a fraction of total thyroid nuclei per field, or Mac2-immunostained cells as a fraction of total thyroid nuclei per field, was performed using Imaris software (version 7.7.2).

Immunocytochemistry for CC3 and β‐catenin was performed to analyse cell death after 24‐hr stimulation with Wnt ± H₂O₂ or to analyse β‐catenin localization after 30 min. VSMCs were fixed with 3% paraformaldehyde/PBS and permeabilized with 0.1%–0.2% Triton X‐100/PBS. After blocking with 20% goat serum/PBS, 1 µg/ml CC3 antibody (AF835; R&D Systems) in 1% BSA/PBS or 2.5 µg/ml β‐catenin antibody (610154; BD Transduction Laboratories, Oxford, UK) in PBS was added overnight at 4°C. Bound antibodies were detected with biotinylated goat anti‐rabbit IgG (B7389; Sigma‐Aldrich) or biotinylated goat anti‐mouse IgG (BA9200; Vector Laboratories, Peterborough, UK) and then DyLight‐488 Streptavidin (SA‐5488‐1; Vector Laboratories) diluted 1:200 in PBS. Coverslips were mounted in ProLong Gold and DAPI (P36931; Invitrogen, Paisley, UK).

Cells were fixed on slides via immersion in 4% paraformaldehyde for 10 min, followed by 3 washes in PBS and incubation with mouse monoclonal anti-LMP1, p52, p50, RelA, or RelB antibodies at room temperature. Next, slides were treated at room temperature with a Cy5-conjugated Affinipure donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) to label anti-p52 and anti-RelA antibodies or a PE-conjugated goat anti-rabbit antibody (Southern Biotech, Birmingham, AL, USA) to label anti-p50 and anti-RelB antibodies. Nuclei were counterstained with ProLong Gold and DAPI (Invitrogen, Carlsbad, CA, USA).

Thyroid glands from mice and rats were immersion-fixed with 10% formalin and processed for paraffin embedding, sectioning, and H&E staining. For immunofluorescence, 6 μm sections were deparaffinized in Citrisolv and an ethanol series, then heated in citrate buffer (12.3 mM, pH 6) for antigen retrieval, and blocked in 1.5% normal goat serum for 30 minutes at room temperature. Primary antibody incubation was performed overnight at 4°C, followed by incubation of Alexa Fluor–conjugated secondary antibodies (Thermo Fisher). After washing, sections were counterstained and mounted with Prolong-Gold and DAPI (Invitrogen). Images were captured in a Nikon A1 confocal microscope. For anti-Ki67 immunohistochemistry, the VECTASTAIN ABC Kit (Vector) was used. After antigen retrieval, sections were treated with 3% H₂O₂, blocked in 1.5% normal goat serum for 20 minutes at room temperature, and incubated with anti-Ki67 antibody for 1 hour at room temperature and biotinylated secondary antibody for 30 minutes, followed by incubation with avidin-HRP. Staining was visualized by DAB reaction. Sections were counterstained with hematoxylin, dehydrated in a graded series of ethanol, and mounted with Permount. Images were obtained with a Leica DMI-3000B microscope.

The expression of CD137 protein on EBV-infected cells was examined by immune-fluorescent staining. Cells were fixed on slides by immersing in 4% paraformaldehyde for 10 min, followed by washing three times in PBS and incubation with mouse monoclonal anti-CD137, goat polyclonal anti-EBNA1) antibodies (Abcam, Cambridge, MA, USA), Cy5-conjugated Affinipure donkey anti-mouse antibody, and FITC-conjugated donkey anti-goat antibody (Jackson ImmunoResearch Laboratories, Inc. PA, USA). Nuclei were counterstained with ProLong Gold and DAPI (Invitrogen, Carlsbad, CA, USA), and the cells were analyzed by confocal microscopy (Fluoview FV10i, Olympus).