3 3 diaminobenzidine (dab)

Immunohistochemistry was used to detect 67LR, macrophages (CD68), T cells (CD3), and fibroblasts (S100A4). After deparaffinization and antigen retrieval, kidney sections were treated with 3.3% H₂O₂ to block endogenous peroxidase activity, and blocked with 5% normal goat serum. The sections were incubated overnight at 4 °C with primary antibodies against 67LR (Abcam, Cambridge, UK), CD68 (Abcam), CD3 (BD Pharmingen, San Diego, CA, USA), and S100A4 (Abcam). The Simplestain MAX-PO (rat) kit (Nichirei, Tokyo, Japan) was used as a secondary antibody and allowed to incubate for 30 min at room temperature^{42 (link)}. Bound antibody was visualized using 3,3'-diaminobenzidine (DAB; Beyotime Biotechnology, Shanghai, China), and nuclei were stained with haematoxylin. The images were captured with a fluorescence microscope (Nikon Ti-E) and the numbers of DAB-positive cells in the 10 random fields for each kidney were counted. The results are expressed as the number of positive cells per square millimetre of renal tissue.

Formalin-fixed paraffin-embedded tissues (FFPE) and tumor microarray (TMA) sections (5 µm thickness) were deparaffinized in xylene and rehydrated in descending alcohols, followed by blocking of endogenous peroxidase in 3% hydrogen peroxide solution (30 min) and heat-induced epitope retrieval in citrate buffer (95 °C, 5 min). Sections were incubated with CD206 (1:100, Clone: C-10, Santa Cruz), iNOS (1:100, Clone: C-11, Santa Cruz), CD16b (1:100, Clone: CLB-gran11.5, BD Biosciences), Ly6G (1:100, Clone: 1A8, BioLegend), p-Smad3 (1:100, Clone: 1D9, Santa Cruz), p-Smad3 (1:200, 600-401-919, Rockland) antibodies overnight (4 °C), sections were incubated in polymer-HRP conjugated secondary antibody (Dako) for 2 h at room temperature, followed by DAB (Thermo-fisher) or Opal-520, 570, 650, 690 TSA dye (Akoya biosciences) development. DAB-stained section images were captured by the Nikon Ni-U Light Microscope and analyzed using Image J analysis software; Opal TSA-stained sections were captured on the Mantra quantitative pathology workstation (Akoya Biosciences) and analyzed by inForm image analysis software 2.6 (Akoya Biosciences) as per our previous studies^{18 (link),51 (link)}.