Halo link software

Blood, liver, and kidney were collected upon euthanasia. Tissues were fixed in 4% paraformaldehyde for 24 hours and then placed in 70% ethanol at 4°C for 4 to 6 days before paraffin embedding. Sections were cut at 5 μm and stained with hematoxylin and eosin on the Leica ST5020 Autostainer, and whole slide scanning was performed by a Leica AT2 slide scanner at ×20 magnification. Slides were reviewed by two veterinary pathologists using HALO Link software (Indica Labs) and Leica DM 3000 LED microscopes.

Tumor tissues were obtained by operation, fixed in formalin and embedded in paraffin. For the Panck/Foxp3/PD-1/CD4/CD8 multiplex panel, a cocktail of primary antibodies including Panck, Foxp3, PD-1, CD4, and CD8 were used, Immunofluorescence staining was performed according to standard procedures (Akoya Biosciences, NEL871001KT). Briefly, paraffin sections were repaired using sodium citrate for 10-25 min, blocked using BF block buffer containing 30%-40% goat serum for 1 hour at room temperature, then incubated with primary antibodies against Panck (Zsgb.bio, catalog number zm0069, 1:200 dilution), Foxp3 (Abcam, catalog number ab20034, 1:100 dilution), PD1 (Zsgb.bio, catalog number zm0381, stock solution), CD4 (Zsgb.bio, catalog number zm0418, stock solution), CD8 (Abcam, catalog number ab199016, 1:500 dilution) at 4°C overnight with additional 2 h at room temperature, and then incubated with secondary antibodies for 10 min followed by appropriate opal fluorophore (690, 520, 570, 480 and 780, respectively) reagent at room temperature for 10min. Finally, the paraffin sections were stained with DAPI (1:500 dilution) for 7 min at room temperature and subjected to standard analysis by Halo Link software (Indica Labs).