Diaminobenzidine dab

For immunohistochemistry, paraffin-embedded hearts were cut transversely into 4- to 5-μm sections. Next, the sections were deparaffinized and blocked with 10% bovine serum albumin. Then, the sections incubated overnight at 4 °C with primary antibodies and anti-rabbit HRP reagent (Gene Tech, Shanghai, China) at 37 °C for 60 min. Finally, the sections were visualized with diaminobenzidine (DAB) (Gene Tech, Shanghai, China) for 2 min at 37 °C and mounted with neutral gums. The sections were examined under a light microscope (Nikon H550L, Tokyo, Japan). For immunofluorescence, the sections were autoclaved for antigen retrieval and then blocked with 10% goat serum for 10 min. Next, the sections were incubated with primary antibodies against CD206 overnight at 4 °C. The sections were rinsed with PBS for 20 min before incubating with two different IRDye® 800CW-conjugated secondary antibodies for 60 min and subsequently counterstained with the SlowFade Gold antifade reagent containing DAPI.

The sections were deparaffinized, rehydrated, immersed in antigen-retrieval buffer, and treated with diluted hydrogen peroxide (0.3%). Then, the sections were incubated with primary antibodies (listed in Table S2) at 4 °C overnight. Subsequently, the sections were stained with horseradish peroxidase (HRP)-labeled secondary antibodies (listed in Table S2) and diaminobenzidine (DAB, Gene Tech, Shanghai, China). Finally, the slides were counterstained with hematoxylin, dehydrated, cleared and, coverslipped. The images were quantified using Image J software (NIH).

Detailed information concerning the tissue microarray (TMA) construction was provided in our previous report 22 (link). Briefly, the TMA included 85 cases of squamous carcinoma, 110 adenocarcinomas and 13 other types of NSCLC. Following dewaxing at 60°C for 2 h and rehydration, the paraffin sections were blocked using 3% H₂O₂ for 15 min to inactivate endogenous peroxidase activity. Subsequently, the sections were incubated in citric acid epitope retrieval reagent at 100°C for 20 min and allowed to cool to room temperature. To block nonspecific binding sites, the sections were treated with 5% bovine serum albumin (BSA) (YESEN, Shanghai, China) for 1 h. Then, the sections were incubated with primary antibodies overnight at 4°C. The primary antibodies were rabbit anti-human RNF38 (25132-1-AP; 1:100 dilution; Proteintech) and rabbit anti-human E-cadherin (EP700Y, 1:500 dilution, Epitomics). The slides were incubated with a horseradish peroxidase (HRP)-labeled secondary antibody (Gene Tech, Shanghai, China) for 1 h at room temperature. After washing off the residual secondary antibody that had not bound the primary antibody, the sections were stained with diaminobenzidine (DAB, Gene Tech, Shanghai, China) and observed under a microscope.