Diaminobenzidine dab

Lung cancer tissue arrays (HLugA180Su07) were purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China) for IHC analysis, including 98 LUAD tissues and 82 para-cancerous normal tissues. The primary antibodies used included antibodies against PLEK2 (ab121131, Abcam, UK), COL1A1 (ab34710, Abcam), and GPX3 (ab104448, Abcam). We deparaffinized the tissue arrays and retrieved antigens. Next, we incubated tissues with the primary antibody at 4 ℃ overnight, washed them with phosphate-buffered saline (PBS), and then incubated them with a biotin-conjugated secondary antibody. After washing, we incubated the sections with horseradish peroxidase (HRP) complex and visualized them using diaminobenzidine (DAB) (Maixin Biotech, Fuzhou, China). The IHC score was independently related to staining intensity and the percentage of positive cells. The staining intensity was divided into four levels: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The proportion of stained positive cells was defined as: 1 (0–25% positive cells), 2 (26–50% positive cells), 3 (51–75% positive cells), and 4 (76–100% positive cells). The staining intensity and the percentage scores were multiplied to obtain the total score. All the IHC results were reviewed by pathologist in First Affiliated Hospital, School of Medicine, Zhejiang University.

Kidney tissues were fixed in 4% paraformaldehyde (Servicebio, Wuhan, China), sliced into 4-μm-thick paraffin-embedded sections and then stained with H&E and PAS. For immunohistochemistry staining, formalin-fixed and paraffin-embedded tissue sections were incubated with primary antibodies against Dll4, activated-Notch1, Epsin1, Clathrin, CD68, F4/80, iNOS, interleukin (IL)-6, and TNF-α (all from Abcam, Cambridge, MA) and then horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature according to the manufacturer’s protocol. Diaminobenzidine (DAB) (Maixin) was used as an HRP-specific substrate.

For immunohistochemistry staining, formalin-fixed and paraffin-embedded tissue sections were incubated with primary antibodies against MTCO1 (ab14705, Abcam), CD68 (ab955, Abcam), or CD3 (ab16669, Abcam) and then analyzed using streptavidin peroxidase detection system (Maixin) according to the manufacturer’s protocol. Diaminobenzidine (DAB) (Maixin) was used as a horseradish peroxidase (HRP)–specific substrate. For immunofluorescence staining, formaldehyde-fixed cells or kidney sections were performed with primary antibodies against RFP (ab62341, Abcam), CD63 (sc5275, Santa Cruz Biotechnology), IL-10 (ab9969, Abcam), KIM-1 (MA5-28211, Invitrogen), CD68 (ab955, Abcam), iNOS (ab15323, Abcam), CD206 (ab64693, Abcam), and CD3 (ab16669, Abcam), followed by incubation with secondary antibodies. Cell nuclei were stained with DAPI. Immunostained samples were visualized under a confocal microscope (FV1000, Olympus).

After conventional dewaxing, coronal sections were treated in 3% hydrogen peroxide (Sinopharm) to eliminate the endogenous peroxidase activity and then blocked in 1% BSA (Sangon Biotech
Co., Ltd.) solution. Next, an anti-rabbit ionized calcium binding adaptor molecule 1 (Iba-1) antibody (1:200, Abcam, Cambridge, UK) was added, and the sections were incubated overnight at
4°C. After being rinsed, the sections were incubated with an HRP-labeled goat anti-rabbit secondary antibody (1:500, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 1 h. The slices
were then developed with diaminobenzidine (DAB, Maixin Biotech. Co., Ltd., Fuzhou, China), and nuclei were counterstained with hematoxylin (Solarbio Life Sciences). Finally, images were
acquired with a light microscope (400× magnification, Olympus).