Ab217344

The tumors were collected from the mice and fixed with 4% paraformaldehyde for 24 h. Samples were dehydrated through a series of graded ethanol baths and then infiltrated with paraffin. Then, 5-µm-thick sections were obtained using a rotary microtome (Leica RM2235, Manual Rotary Microtome), rehydrated and permeabilized in 0.5% Triton X-100 (dissolved in PBS; catalog no. 9002-93-1; Sigma) for 20 min. Nonspecific staining between the primary antibodies and the tissue samples was blocked by incubating sections in the block buffer (1% fetal bovine serum in PBS) for 1 h at room temperature. After incubating with the anti-mouse CD8α antibody (1:500; catalog no. ab217344; Abcam) overnight at 4 °C, the slides were washed three times for 15 min each time in PBS and then incubated with the Alexa Fluor 488 goat anti-rabbit immunoglobulin G antibody (1:1000; catalog no. ab150077; Abcam) for 40 min at room temperature. After washing three times with PBS, the slides were incubated with DAPI solutions (5 µg/ml; catalog no. Cl002; Beyotime Inc.) for 10 min at room temperature. The slides were further washed three times with PBS, mounted in the antifade mounting media, and imaged by a fluorescence microscope (DMI8; Leica) equipped with an Olympus digital camera (Olympus DP71; Olympus). CD8α-positive cytomembrane was stained green, and all nuclei were stained blue.

Immunohistochemical staining was performed as previously reported^{56 (link)} with the following antibodies: anti-insulin (dilution 1:100, #4590; Cell Signaling Technology), anti-PDX-1 (dilution 1:50, #5679; Cell Signaling Technology), anti-PD-L1 (dilution 1:100, 17952–1-AP, Proteintech), anti-ICA (dilution 1:500, ab207750; Abcam plc.), anti-ZnT8 (dilution 1:500, 16169–1-AP, Proteintech), anti-GAD65 (dilution 1:2000, ab239372; Abcam plc.), anti-CD4 (dilution 1:1000, ab183685; Abcam plc.) and anti-CD8 (dilution 1:2000, ab217344; Abcam plc.).

The dissected tumours were dewaxed and hydrated, after blocking the non-specific sites by 5% BSA, the tissues were incubated with primary antibody DSN1 overnight at 4 °C. After washing the tissues were overlaid with appropriate secondary antibody for 2 h at room temperature, then detected using DAB reagent.
The tumours were fixed in 4% paraformaldehyde and cut into 4-μm sections. Before staining, all sections were subjected to antigen retrieval and blocking of endogenous peroxidase. Slides were incubated with the primary antibodies Ifn-γ (15365-1-AP, Proteintech, 1:200 dilution), Cd8 (ab217344, Abcam, 1:2000 dilution), Pdl-1 (66248-1-Ag, Proteintech, 1:10000 dilution), Cxcl9 (701117, Invitrogen, 1:200 dilution) overnight, and the rabbit or mouse secondary antibody at room temperature for 1 h. DAB was added to visualize the antigens in situ. The hematoxylin and eosin staining was performed according to the established protocols.²⁵ (link)

The sorting of CD4⁺ T cells or CD8⁺ T cells were labeled with 10 μM carboxyfluorescein succinimidyl ester (CFSE) (Thermo CellTrace™ CFSE Cell Proliferation Kit C34554) for incubation at room temperature in the dark for 10 mins. The cells were centrifuged and then washed three times with Roswell Park Memorial Institute 1640 (RPMI 1640) containing 10% FBS. CD4⁺ or CD8⁺ T cells were seeded in 24-well plates (5 × 10⁵ per well) and co-cultured with magnetic beads (Miltenyi, T-cell activation/expansion kit, 130–093–627) of the same proportion in RPMI 1640 supplemented with 10% FBS and IL-2 (50 U/mL) according to the manufacturer’s protocol. After 48 h, 96 h, and 144 h, CD4⁺ T-cell and CD8⁺ T cells were partially harvested and stained with anti-CD4 (1:100, ab133616, Abcam) or anti-CD8a (1:500, ab217344, Abcam). Proliferation was detected by FACSAria II cell flow cytometer (BD Biosciences, CA, USA). The data were analyzed with FlowJo softwar.

Lungs were collected from indicated mice sacrificed on day 6 post-infection. Tissues were covered in cryo-embedding media, kept in liquid nitrogen until completely submerged, then stored at −80 °C until ready for sectioning. 4 mm sections were cut with a cryotome and stained with haematoxylin and eosin. For immunofluorescence, sections were fixed in 4% paraformaldehyde for 1 h at RT, washed with PBS, then incubated in “permeabilisation solution” containing 0.05% TX-100 and 0.1% sodium citrate for 2 min on ice (4 °C). Cell death was analyzed with an in situ cell death detection kit (TMR-red, Roche) after antigen retrieval either alone or followed by staining with anti-CD45 (AB 10558 Abcam) and Goat anti-Rabbit IgG DyLight488 (ThermoFicher ref: 35553). Anti-CD8a (AB 217344, Abcam) was used 1/100 combined with a secondary Goat anti-Rabbit IgG AF568. Hoechst 33342 was used to stain nuclei. Brightfield and fluorescence microscopy was performed using ZEISS Axio Scan.Z1. Samples were analyzed with Zen 3.2. blue edition (Zeiss) and quantified with QuPath software 0.2.3.