Metamorpho version 6

Immunohistochemistry was performed as described previously [14 (link)]. Paraffin-embedded tissues were cut into sections 4-µm thick, mounted on glass slides, and stained using indirect immunoperoxidase. The slides were processed for immunodetection of transforming growth factor β (TGF-β) and angiotensin II type 1 receptor (AT1R) with antibodies specific for TGF-β (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-AT1R (Santa Cruz Biotechnology) respectively. Diaminobenzidine (Sigma Chemical Co., St. Louis, MO, USA) was used as a chromogen. All samples were evaluated under an Olympus BX51 microscope. The size of the area stained positively for TGF-β (as a percentage of the total area in 10 separate fields of each section under ×200 magnification) was determined using a digital camera-based image analyzer (Metamorpho version 6.3, Olympus).

Tissue staining was performed as described previously [14 (link)]. Briefly, paraffin-embedded kidney pieces were cut into sections 4-µm thick and mounted on glass slides. The sections were deparaffinized with xylene, stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT), and examined under an Olympus BX51 microscope (Olympus, Tokyo, Japan). The tubulointerstitial injury score was evaluated based on morphological changes in tubuels, such as dilatation, distortion of tubular basement membranes and atrophy: grade 0, no morphological deformities; grade 1, less than 10%; grade 2, less than 25%; grade 3, less than 50%; grade 4, less than 75%; and grade 5, 75% or greater involved. Ten fields of the outer medulla were evaluated. Renal fibrotic areas were quantified using morphometric analysis with blue stained area in MT staining. The size of the blue-stained (fibrotic) area in MT staining (as a percentage of the total area in 10 different fields of each section under ×200 magnification) was determined using a digital camera-based image analyzer (Metamorpho version 6.3, Olympus).