*4.4. Histological Analysis of the Kidneys*

The kidney tissue was fixed with 4% phosphate-buffered formalin solution (Macron Chemicals, Center Valley, PA, USA), embedded in paraffin block and cut into 4-μm sections. For histological analysis, after deparaffinization and rehydration by xylene and graded alcohols, the sections were subjected to Masson trichrome staining according to the manufacturer's instructions (Accustain, Sigma-Aldrich). Twenty randomly selected nonoverlapping high-power fields (40× objective) were evaluated for each mouse, and the average for each group was then analyzed. The fibrotic area was quantified by ImageJ software (1.52a, US National Institutes of Health, Bethesda, MD, USA).

#### *4.5. Immunohistochemical Staining*

Immunohistochemical staining was performed on formalin-fixed paraffin-embedded sections of obstructed kidneys, as previously described [27]. Briefly, after deparaffinization by xylene and rehydration by graded alcohols, consecutive 4-μm sections of kidneys were subjected to heat antigen retrieval in a microwave oven (650W, 12 min) in a 10-mM sodium citrate buffer (pH 6.0). Afterwards, endogenous peroxidase activity was quenched by 3% hydrogen peroxide (Sigma-Aldrich) for 10 min. Thereafter, tissue sections were incubated with the primary antibodies at 4 ◦C overnight and then with the secondary antibody (Envision+Dual Link System-HRP, Dako, Glostrup, Denmark) for 30 min at room temperature. Signals were developed with diaminobenzidine substrate-chromogen (DAB, Dako), which resulted in a brown-colored precipitate at the antigen site. Finally, sections were counterstained with a Gill's hematoxylin (Merck, Darmstadt, Germany). Primary antibodies included F4/80 (1:100, Cat#sc-25830, Santa Cruz Biotechnology, Santa Cruz, CA, USA), iNOS (1:200, Cat#sc-651, Santa Cruz Biotechnology), Arg1 (1:200, Cat#sc-20150, Santa Cruz Biotechnology), α-SMA (1:200, Cat#ab5694, Abcam, Cambridge, UK), CLEC7A (1:50, Cat#TA322197, OriGene Technologies, Rockville, MD, USA) and SLAM (1:100, Cat#ab156288, Abcam). Twenty randomly selected nonoverlapping high-power fields (40× objective) at the renal cortex were evaluated for each mouse. Analysis of the

DAB-positive area was carried out using Image J with the "Threshold Colour" plug-in (version 1.16, https://imagejdocu.tudor.lu/plugin/color/threshold\_colour/start#threshold\_colour).
