Immpact dab

IHC was performed on formalin-fixed paraffin-embedded tissue sections. Following dewaxing, rehydration, and endogenous peroxidase blocking by a 3% solution of H2O2 (Sigma) for 20 min. Antigen retrieval was performed using citrate or Tris-EDTA buffers as per antibody datasheets. Nonspecific antibody binding was blocked by incubation with either horse or goat serum (Vector). Primary antibodies were diluted 1:500 (PD1, FOXP3) or 1:200 (CD4, CD8) in Antibody dilution buffer (Life Technologies #003218) and were applied for 1h at room temp. Following two washes in TBS, ready-to-use secondary antibodies were applied (ImmPRESSTM HRP reagent kits, Vector). Sections were washed twice in TBS and ImmPACTTM DAB (Vector) was used to detect immunolabeling. Sections were counterstained in hematoxylin, and following dehydration and clearing in xylene, were mounted in distyrene/plasticizer/xylene. Sections were scanned using Aperio and Imagescope software (Leica Biosystems) was used to determine percentage positive cells.

The formalin-fixed liver tissue samples were immunostained for CD68 and α-smooth muscle actin (SMA). Tissue sections were deparaffinized and unmasked using Histo/Zyme (Dagnostic BioSystems, Pleasanton, CA). They were subsequently washed using phosphate-buffered saline (PBS) and a solution containing H₂O₂ to block endogenous peroxidase activity. The sections were incubated overnight with anti-CD68 (Abcam plc, Cambridge, UK) or anti-α-SMA (Abcam plc, Cambridge, UK) antibodies; this was followed by their incubation with the secondary antibody MAX-PO (MULTI) (Nichirei Biosciences Inc., Tokyo, Japan). After washing with PBS, color was developed using peroxidase substrate ImmPACT^TM DAB (Vector Laboratories, Burlingame, CA) and observed by light microscopy using DM750.

Sections were rinsed in 0.1M PBS, then endogenous peroxidase activity was blocked using 10:1 methanol/30% hydrogen peroxide for 20 min in the dark. After rinsing with PBS, sections were blocked for nonspecific antigen binding using 4% BSA/0.3% Triton X-100/PBS (BP³⁺) for 1 h in a humid chamber. Primary antibodies (Table 1) were diluted in BP³⁺ and applied to sections overnight at room temperature. Next, sections were rinsed and treated with biotinylated secondary antibodies for 1 h at room temperature. Sections were primed with Elite avidin-biotin enzyme complex (ABC; Vector Laboratories) for 1 h, and labeling was visualized with ImmPACT^TM DAB (Vector Laboratories). Sections were rinsed, dehydrated, and coverslipped with Permount (Thermo Fisher Scientific).

Colon tissue sections were deparaffinized by using xylene series and hydrated through graded ethanol series (100%, 95%, 70%). After rinsing in tap water, tissue sections were incubated in a hydrogen peroxide blocking solution for 10 minutes. The sections were then washed with PBS and incubated for 1 hour in a working solution of anti M.O.M.^TM Mouse Ig Blocking Reagent (Vector Laboratories, Burlingame, Calif., USA). Tissue sections were again washed in PBS and then incubated in ready-to-use 2.5% normal horse or goat serum (Vector Laboratories) for 30 minutes. Sections were then incubated overnight with the following primary antibodies: WIF-1, SSTR-1 (somatostatin receptor 1), group II PLA2g2 (phospholipase A2) and CD31 (Santa Cruz Biotechnologies, Santa Cruz, Calif., USA), neurotensin (Novus Biologicals, Littleton, CO), PCNA (proliferating cell nuclear antigen) and MECA-32 (Biolegend, San Diego, CA). Sections were then washed with PBS the next day and incubated for 30 minutes in the anti-rabbit, anti-rat or anti-mouse M.O.M.^TM ImmPRESS^TM Reagent (Vector Laboratories). Tissues were again washed in PBS and then incubated in a peroxidase substrate solution, ImmPACT^TM DAB (Vector Laboratories) as chromogen.

Pancreatic cancer TMA slide containing 82 pancreatic cancer and 54 normal samples was purchased from OUTDO BIOTECH (Shanghai, China). The slide was deparaffinized, rehydrated, and antigens were retrieved by Antigen Retrieval Citra Plus Solution (BioGenex, #HK080-9K), followed by treatment with 3% H₂O₂ to block endogenous peroxidases. After primary IGF2BP2 antibody, and secondary antibody, and ABC staining using VECTASTAIN^® Elite^® ABC HRP Kit (VectaStain, #PK-6101), the signals were revealed by ImmPACT^TM DAB (Vector Laboratories, #SK-4105). The overall IGF2BP2 expression was calculated from total score (signaling intensity score x staining distribution score). The signal intensity scores were: 0 (no signal), 1 (weak), 2 (moderate), and 3 (strong). The staining distribution scores were based on the percentage of positive cells: 0 (0%), 1 (1–10%), 2 (10–50%), and 3 (51–100%). Clinical and histopathological information of patients were shown in Supplementary Table 2.

The heart and liver tissues were fixed in 10% (w/v) neutral buffered formalin and embedded in paraffin, and 4 μm-thick sections were prepared. The sections were deparaffinized with xylene, and endogenous peroxidase activity was blocked with hydrogen peroxide. Next, the cells were washed with phosphate buffer and treated overnight with anti-CD68 (Abcam plc, Cambridge, UK) or anti-p47phox (Santa Cruz Biotechnology, Inc., Dallas, CA, USA) antibodies. They were then treated with the secondary antibody MAX-PO (MULTI) (Nichirei Biosciences Inc., Tokyo, Japan). Color was developed using the peroxidase substrate diaminobenzidine (ImmPACT TM DAB; Vector Laboratories, Inc., Burlingame, CA, USA) [52 (link)]. Immunostaining was quantified by randomly imaging CD68 (ten fields of liver tissue at 100×) or p47phox (20 fields of liver tissue at 400×) staining using an optical microscope equipped with a high-resolution video camera (CX41; Olympus, Tokyo, Japan). Next, the ratio (%) of the positively stained region (brown area) to the total area of the liver tissue was calculated. All images were analyzed using the ImageJ software (version 1.52, National Institutes of Health, New York, NY, USA).

IHC was performed on formalin-fixed paraffin-embedded tissue sections, following dewaxing, rehydration and endogenous peroxidase blocking by a 3% solution of H2O2 (Sigma) for 20 min. Antigen retrieval was performed using citrate or Tris–EDTA buffers as per antibody datasheets. Nonspecific antibody binding was blocked by incubation with either horse or goat serum (Vector). Primary antibodies were diluted 1:500 (PD-1) or 1:200 (CD8) in Antibody dilution buffer (Life Technologies #003218) and were applied for 1 h at room temp. Following two washes in TBS, ready-to-use secondary antibodies were applied (ImmPRESSTM HRP reagent kits, Vector). Sections were washed twice in TBS, and ImmPACTTM DAB (Vector) was used to detect immunolabelling. Sections were counterstained in hematoxylin and, following dehydration and clearing in xylene, were mounted in distyrene/plasticizer/xylene. Sections were scanned using Aperio, and ImageScope software (Leica Biosystems) was used to determine percentage positive cells.