Epr19514

Four-millimetres punch biopsies from the ear were fixed in 4% formaldehyde overnight at 4°C, paraffin-embedded, and serially sliced into 4 µm sections subjected to staining procedures. For haematoxylin & eosin (HE) staining, a standard protocol was followed. To detect mast cells, sections were stained with 0.1% toluidine blue solution (Sigma-Aldrich) at pH 2.3 and room temperature for 3 minutes. For immunohistochemical staining, heat-induced antigen retrieval was performed (25 minutes at 97° C) in Tris-EGTA buffer (pH 9), and stained with anti-CD4 antibody (1:1000; EPR19514, Abcam, Cambridge, UK) or anti-Ly6g antibody (1:2000; EPR22909-135, Abcam) for 1 hour at room temperature. The remaining steps for detection were performed with Ultravision Quanto Detection System (ThermoFisher) following the manufacturer’s instructions. The sections were counterstained with hematoxylin.
All slides were scanned with 20× objective using the whole slide scanner NanoZoomer 2.0-HT (Hamamatsu Photonics K.K, Hamamatsu City, Japan). Quantitative image analysis was performed in QuPath 0.4.2 using the Cell Detection command with default settings followed by a manual review and corrections (26 (link)). The number of counted cells was normalised to the length of the section

For multiple fluorescence immunohistochemistry, Liu et al. can be referred to (26 (link)), and a four-color multiple fluorescence immunohistochemistry staining kit (Absin, Shanghai, China) was used. Briefly, the slices were processed for heat-mediated antigen retrieval by using citric acid buffer. The sections were blocked with goat serum, and then primary antibody was added and incubated overnight at 4°C. On the next day, the sections were washed with TBST and incubated with a second antibody for 15 min, then washed with TBST, and incubated with tyramine signal amplification (TSA) monochrome fluorescent dye for 10 min, after which the above-mentioned steps were repeated. Finally, 4′6-diamino-2-phenylindole (DAPI) was used for nuclear staining. All images were acquired by using Nikon A1 plus a laser confocal microscope. The primary antibodies used for immunofluorescence were anti-CTLA4 antibody (CAL49; 1:500, Abcam), anti-CD4 antibody (EPR6855; 1:500, Abcam), anti-CD4 antibody (EPR19514; 1:1,000, Abcam), anti-CD8 alpha antibody (EPR21769; 1:2,000, Abcam), and anti-CD8 alpha antibody (EPR22483-288; 1:1,000, Abcam).

Paraffin-embedded sections of mice brains were immunostained for antibodies specific to adenoviral mouse-hexon (1:1000; AB1056, Millipore), adenovirus rabbit-E1A, (1:1000; Santa Cruz Bio-technology, Santa Cruz, CA), CD3 (1:300; clon SP7, NeoMarkers, Fremont, CA), CD4 (1:1000; EPR19514, ab183685 Abcam, Cambridge, MA) CD8a (1:1000, (D4W2Z) #98941 Cell Signaling, Danvers, MA) and FoxP3 (1:400; clon JFK-16s, ref. 14–5773 eBiosciences, Thermo Fisher, Waltham, MA) following conventional procedures. For the immunohistochemical staining, Vectastain ABC kits (Vector Laboratories Inc., Burlingame, CA) were used according to the manufacturer’s instructions.

Tumors were formalin-fixed and paraffin-embedded and processed into 4 μm tissue sections. Tumor sections were stained for FAP (EPR20021, Abcam), CD8 (D4W2Z, Cell Signaling), and CD4 (EPR19514, Abcam) on a Bond Rx autostainer (Leica Biosystems) with Bond Polymer Refine Detection (Leica Biosystems) according to the manufacturer's protocol. After staining, sections were dehydrated and film cover slipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2 (Leica Biosystems). Image analysis and quantification was performed on Aperio ImageScope v12.1 (Leica Biosystems) or QuPath v0.1.2 [18 (link)].

For histology, livers were fixed in 10% formalin, embedded in paraffin, and sliced into 5-µm sections. Hematoxylin and eosin (H&E) staining was then conducted on sections. Confluent liver necrosis was measured on H&E-stained liver sections via ImageJ software (edition 1.53c, National Institutes of Health, USA). Immunohistochemistry was performed using antibodies for CD4 (EPR19514, Abcam, USA). The TUNEL assay was conducted using the One-step TUNEL FITC Apoptosis Detection Kit (APPExBIO, USA) according to the instructions.