Dab solution

Immunohistochemical staining was performed using a commercial kit (BOSTER, China), Briefly, tumor tissue slices were dewaxed with ethanol and xylene, incubated in citrate buffer for antigen repair, blocked with BSA for 1 h, and then incubated with rabbit NASP pAb (11323‐1‐AP; Proteintech, 1:100), mouse annexin A2 mAb (66035‐1‐Ig; Proteintech, 1:5000), rabbit phospho‐annexin A2 (Tyr23) pAb (AF7096; Affinity, USA, 1:100), rabbit STAT3 pAb (10253‐2‐AP; Proteintech, 1:200), and rabbit phospho‐STAT3 (Y705) pAb (EP2147Y; Abcam, UK, 1:100) at 4°C overnight. The slices were then washed three times with PBS, incubated with anti‐rabbit or anti‐mouse secondary antibodies, washed three times with PBS, and stained with DAB solution (Service Bio, China) under radiotherapy for 5–10 min. Images were captured using a light microscope (Nikon, Tokyo, Japan).

Immunohistochemistry analysis was performed as previously described [38 (link)]. Briefly, the formalin-fixed paraffin embedded mouse and human PDAC tissue blocks were subjected to immunohistochemical staining using primary rabbit anti-F4/80 (1:100; Proteintech, USA), rabbit anti-CD68 (1:100; Proteintech, USA) and rabbit anti-TGF-β (1:100; Servicebio, China) antibodies, followed by incubation in secondary biotinylated antibody and finally visualized with DAB solution (Servicebio, China) and counterstained with hematoxylin (Servicebio, China). Picture were taken under a light microscope (Olympus BX50, Japan).

The retinal frozen sections were treated with 30% hydrogen peroxide solution to remove pigment and soaked in PBS 3 times for 2 min each. The sections were then incubated with Perls reagent (5% potassium ferrocyanide (Sigma, P3289) and 5% HCl in ddH₂O) for 1 h at 25 °C. After removing the Perls reagent, the sections were washed with ddH₂O 3 times for 2 min each. Sections were treated with DAB solution (Servicebio, China) for 30 min to amplify the signals, and then washed with ddH₂O 3 times for 5 min each. Finally, sections were mounted with 90% glycerol in PBS (PH 7.6) and imaged with optical microscope BX53.

After deparaffinization and rehydration, distal colon sections were microwaved in citric acid (pH 6.0). After washing with phosphate-buffered saline containing Tween-20 (pH 7.4), the sections were incubated in a 3% H₂O₂ solution and blocked with 3% bovine serum albumin for 20 min at room temperature (Zhao et al., 2020 (link)). The sections were then incubated with NF-κB p65 (1:100; CST, Danvers, MA, United States), LC3 (1:200; CST, Danvers, MA, United States), and Beclin 1 (1:150; CST, Danvers, MA, United States) primary antibodies at 4°C overnight. The blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:200; Servicebio, China) for 30 min at room temperature, followed by DAB solution (Servicebio, China). The sections were counterstained with hematoxylin, dehydrated, and mounted. Sections were observed using Caseviewer and Leica LAS image acquisition systems. Positive DAB staining areas were quantified using the NIH ImageJ software (National Institutes of Health, Bethesda, MD, United States).

Paraffin sections of lung tissue were first dewaxed to water, and then antigenic repair was performed to block endogenous peroxidase. After serum sealing, primary antibodies MMP9 antibody (AF5228; Affinity, Cincinnati, OH, USA), and vascular cell adhesion molecule-1 (VCAM-1) antibody (DF6082; Affinity) were added. Subsequently, horseradish secondary rabbit peroxidase antibody (HRP) (AF5131; Abcam, Cambridge, UK) and 50 mM Tris–HCl buffer (pH 7.4) were added and incubated. It was further exposed to DAB solution (G1212-200T; Servicebio) for 15 min and reacted with hematoxylin for 3 min.

For immunohistochemistry, deparaffinised sections were boiled in sodium citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase activity was inhibited by 3% hydrogen peroxide, and nonspecific binding was blocked using bovine serum albumin (BSA). Sections were then incubated overnight with the primary antibodies (Ki67: 1:750, #GB111141, Servicebio; CD31: 1: 600, #GB11063-2, Servicebio) at 4 °C. After incubation, sections were washed and incubated with the secondary antibody, HRP-conjugated goat anti-rabbit IgG (1:200, #G1213, Servicebio) at 23–25 °C for 50 min. Immunostaining was developed using a DAB solution (#G1211, Servicebio).
For immunofluorescence, deparaffinised sections were boiled for antigen retrieval and blocked for non-specific binding with BSA. The sections were then either incubated with fluorescent avidin conjugates (#A21370, Invitrogen) for 1 h at 23–25 °C for the identification of MCs, or overnight with the primary antibody at 4 °C (IL-33: 1:100, #AF3626, R&D Systems). After overnight incubation, sections were washed and incubated with the secondary antibody, Cy3-conjugated donkey anti-goat IgG (1:200, # GB21404, Servicebio), for 1 h at RT. Nuclei were stained with DAPI (#GDP1024, Servicebio). Fluorescence images were captured using a microscope (Nikon; Tokyo, Japan).

Immunofluorescence staining and immunohistochemistry staining were performed following standard procedures. For immunofluorescence staining, bone sections were incubated with anti‐SOST antibody (#21933‐1‐AP, Proteintech, China, 1:100) at 4°C overnight and washed with PBS for three times, and incubated with the secondary goat anti‐mouse IgG H&L antibody (1:400) for 30 min. Nuclei were stained with DAPI. The images were captured by a fluorescence microscope (Nikon, Japan). For immunohistochemistry staining, sections were submerged with sodium citrate at 55°C overnight for antigen retrieving. After being blocked with BSA for 1 h, the sections were incubated with anti‐osteocalcin antibody (#23418‐1‐AP, Proteintech, China, 1:100) at 4°C overnight and washed with PBS for three times and incubated with anti‐rabbit secondary antibody (ZsBio, China). Sections were washed with PBS for three times and stained with DAB solution (ServiceBio, China) at RT for 5–10 min. The images were captured by microscope (Nikon, Japan).