Hrp polymers

Harvested tissues were immediately fixed in 10% formalin overnight and embedded in paraffin. IHC was performed as described previously^{62 (link)}. Briefly, endogenous peroxidases were inactivated by 3% hydrogen peroxide. Non-specific signals were blocked using 3% BSA, 10% goat serum in 0.1% Triton X-100. Tumor samples were stained with the following primary antibodies: IL2Rγ (Abcam ab180698, Bioss bs-2545R); Ki67 (Vector Laboratory, VP-RM04); IL4R (Bioss bs2458R); cMyc (Abcam ab32072); Hexokinase II (Abcam ab209847); Gata-3 (CST 5852); LDHA (CST 3582); IL13rα1 (Abcam ab79277); Jak1 (CST 3344); pSTAT1-Y701 (CST 9167); pSTAT3-Y705 (CST 9145); Stat5 (CST 94205); Stat1 (CST 9172); IL4 (Abcam ab9622); IL13 (Abcam ab106732); CD45 (Abcam ab10558), CD4 (Abcam ab183685) and F4/80 (Abcam ab6640). After overnight incubation, the slides were washed and incubated with secondary antibody (HRP-polymers, Biocare Medical) for 30 min at room temperature. The slides were washed three times and stained with DAB substrate (ThermoFisher Scientific). The slides were then counterstained with haematoxylin and mounted with mounting medium. For clinical samples, staining intensity of tissue sections was scored in a ‘blinded’ manner by two independent pathologists.
Immunofluorescence slides were imaged with an Olympus Microscope and quantified with ImageJ.

Tumors were fixed in formalin for 24 h, paraffin embedded, sectioned, and stained according to standard procedures. Briefly, endogenous peroxidases were inactivated by 3% hydrogen 673 peroxide. Non-specific signals were blocked using 3% BSA, 10% goat serum in 0.1% Triton X-100. After antigen retrieval in citrate buffer, slides were stained using respective antibodies overnight at 4°C, [ ENO1 (Proteintech, #11204–1-AP), PGK1 (Proteintech #17811–1-AP), PGAM1 (Proteintech, #16126–1-AP), Tyrosinase (Abcam, ab738), pAKT (Cell Signaling, #9271), pIGF1R (Abcam, ab39398)]. After overnight incubation, the slides were washed and incubated with secondary antibody (HRP-polymers, Biocare Medical) for 30 min at room temperature. The slides were washed three times and stained with DAB substrate (ThermoFisher Scientific). The slides were then counterstained with hematoxylin and mounted with mounting medium.