Masson s trichrome stain

Paraffin-embedded kidney sections were used for immunohistochemical staining. Matrix deposition within the interstitium was assessed using Masson’s trichrome stain (American MasterTech, Lodi, CA). Briefly, endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide. After pre-incubation with 10% protein block (Dako, CA) for 10 minutes at room temperature to block nonspecific binding of antibodies, the tissues were incubated overnight at 4°C with primary antibodies against KCa3.1, CD68, F4/80, type I collagen, fibronectin, TGF-β1 and p-Smad2/3. After incubation with appropriate secondary antibodies, sections were developed with 3, 3-diaminobenzidine (Dako, CA) to produce a brown colour and counterstained with haematoxylin. Positive signals in the renal cortex regions were quantified using Image J software as previously described [11 (link)]. The number of cells positive for CD68+, F4/80+, phospho-Smad2/3+ were counted in 10 high power fields (HPF, 40×) of the tubulointerstitium.