Rat anti cd8a

YUMM1.7 and YUMM1.7-CM cells were cultured in black-wall 96-well plates. After treatments, cells were fixed with 50 µl 4% PFA (20 min at 25°C), permeabilized/blocked for 30 min in 50 µl 5% goat serum/0.1% Triton X-100 in PBS, and incubated 2 h with primary antibodies (listed below). Following three washes with PBS/0.1% Tween-20, respective secondary antibodies (1:1,500 Alexa-488/594, Life Technologies) were applied for 30 min. For immunofluorescent analyses of tumoral sections, tumors were excised, snap frozen, embedded in Tissue-Tek OCT (#4583, Sakura Finetek), and cryo-sectioned at 10 μm. Cryosections were permeabilized by heat in citrate buffer (Dako S1699) for 18 min, blocked in PBS with 10% goat serum/0.3M glycine 45 min, and incubated 2 h with primary antibodies in PBS/1.5% goat serum: rat anti-CD8a (1:100, #100702, Biolegend), rat Granzyme B (1:100, ##488898-82, Invitrogen), rabbit Ki-67 (1:200, D3B5, #12202S, Cell Signaling), or rabbit Pfkfb3 (1:300, ab135820, Abcam). After washes with PBS/0.3% Tween-20, sections were incubated 45 min with anti-rabbit or anti-rat IgG dye conjugated antibodies Alexa-488/594 (Life Technologies), mounted with anti-fade reagent with DAPI, and observed with 10 or 40× objective using Olympus IX71 microscope equipped with QIC-F-M-12-C cooled digital camera (QImaging, Surrey, BC) with QCapture Pro (QImaging) software.

After exsanguination, the mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, and the pancreas, thymus and bone were collected. Rabbit anti-insulin (Cell Signaling Technologies, Danvers, MA USA), rabbit anti-glucagon (Cell Signaling Technologies) and rabbit anti-TNF-α (Abcam, Cambridge, UK) were used as primary antibodies, followed by ImmPRESS reagent anti-rabbit IgG as the secondary antibody. The color was developed using an ImmPACT DAB kit (Vector Laboratories, Burlingame, CA, USA). For immunofluorescence analysis, the sections were incubated with anti-Vcam-1 (Cell Signaling Technologies) or rabbit anti-NG2 (Proteintech, USA) and sheep anti-von Willebrand Factor (Abcam) primary antibodies at 4 °C overnight, followed by Alexa555 anti-rabbit IgG as a secondary antibody (Thermo Fisher Scientific Inc. Waltham, MA, USA). To examine the localization of CD8a T cells in the thymus, frozen sections of fixed thymus were stained with rat anti-CD8a (Clone 53-6.7, Biolegend) as primary antibody and Alexa488 anti-rat IgG (Thermo Fisher Scientific) as secondary antibody at 4 °C overnight. These fluorescent immunostained sections were mounted with a DAPI-containing medium (Vector Laboratories) and observed under a fluorescence microscope.