Mouse adsorbed peroxidase polymer detection kit

Paraffin-embedded liver/tumor tissue sections were deparaffinized, rehydrated, followed by heat-induced antigen retrieval and then incubated with primary antibodies as listed in Additional file 1: Table S1. ImmPRESS anti-Rat Ig, ImmPRESS anti-Rabbit Ig, Mouse adsorbed (peroxidase) Polymer Detection Kit, DAB Peroxidase Substrate Kit and Hematoxylin (all from Vector laboratories, Burlingame, USA) were used for detection and visualization. For detection of CD45.1⁺ adoptively transferred CD8⁺ T cells, 5–7 μm of liver/tumor tissue frozen sections were fixed in ice cold acetone/chloroform (1:1), incubated with FITC-conjugated anti-CD45.1 (A20, Biolegend) and anti-FITC conjugate HRP (Thermo Fisher Scientific, Waltham, USA), followed by incubation with DAB substrate. For detection of lipid droplets, 5–7 μm liver/tumor tissue frozen sections were fixed in 10% formalin, incubated with oil red O dye for 15 min and washed with 60% isopropanol to remove the excess dye before mounting with glycerol jelly mounting medium. The images were captured using an automatic digital slide scanner Pannoramic MIDI with Plan-Apochromat 20x/0.8 objective (3D HISTECH) by Pathology Core Laboratory of NHRI and have been evaluated by an experienced pathologist (SFH).

Five-μm section of frozen liver or tumor tissues were fixed in ice-cold acetone for 6 min. Gr-1 (RB6-8C5) and CD11b (M1/70) were purchased from BioLegend. The staining was performed using ImmPRESS Anti-Rat Ig, Mouse Adsorbed (peroxidase) Polymer Detection Kit, DAB Peroxidase Substrate Kit and Hematoxylin (Vector Laboratories, Burlingame, CA, USA). The slides were mounted with VectaMount Permanent (Vector Laboratories). The image was captured using an automatic Digital Slide Scanner Pannoramic MIDI with Plan-Apochromat 20x/0.8 objective (3D HISTECH, 3DHISTECH Ltd., Budapest, Hungary) by Pathology Core Laboratory of NHRI.