Bio revo imaging and analysis software

For renal histology, mouse kidneys were fixed in 10% formalin and embedded in paraffin for Periodic Acid-Schiff (PAS), H&E staining and Masson-Trichrome (MT) staining. Tissue blocks were sliced into 4-μm thickness. Glomerular injury score, inflammatory cell infiltration, and tubulointerstitial fibrosis score were assessed as described previously [12 (link)]. Briefly, for assessment of glomerular injury, renal sections were stained with PAS. To evaluate the glomerular injury score, more than 50 PAS-stained random glomeruli per mouse (n = 4 mice) were examined, and scored from 0 to 4 (0, no lesion; 1, expansion of mesangial area; 2, expansion of Bowman’s epithelial cells and adhesion of glomeruli and Bowman’s capsule; 3, sclerotic area in 50% - 75% of glomerulus; 4, sclerotic area in 75% - 100% of glomerulus). Double blind scoring was performed and values were computed and presented in a graph as percentage. For tubulointerstitial fibrosis score, MT-stained kidneys were evaluated using Bio-Revo imaging and analysis software (Keyence, Japan). MT-stained area vs unstained area was calculated and presented as % fibrotic region.

For histological analysis, mouse renal tissues were fixed in 10% formalin, followed by 70% ethanol dehydration, embedded in paraffin and sectioned at 4-µm or 2-µm thickness for periodic acid-Schiff (PAS), periodic acid-methenamine silver (PAM) and Masson-trichrome staining. Glomerular injury score was assessed as described previously [15 (link)]. More than 100 PAS-stained random glomeruli per mouse (n = 5–6 mice) were examined, and scored from 0 to 4 (0, no lesion; 1, expansion of mesangial area; 2, expansion of Bowman’s epithelial cells and adhesion of glomeruli and Bowman’s capsule; 3, sclerotic area in 50–75% of glomerulus; 4, sclerotic area in 75–100% of glomerulus). Scoring was performed using Bio-Revo imaging and analysis software (Keyence, Japan). Values were computed and presented in a graph as percentage.

For immunohistochemistry, kidney tissues were immersed consecutively in 10% formalin and 75% ethanol, and embedded in paraffin. De-paraffinization step was done with xylene and ethanol. Antigen activation was performed using Dako proteinase K for 15 min or Dako real target retrieval solution (Dako, Japan) at 98°C for 40 min, then samples were reacted with F4/80 (#6640, Abcam) or type Ⅳ collagen (#6586, Abcam) diluted at 1:100. Histofine Simple stain (Nichirei Biosciences Inc., Japan) was used for secondary antibody reactions. After TBS wash, DAB reaction was performed for 1–10 min. Slides were stained with Haematoxylin for 60 sec. F4/80 and type Ⅳ collagen were evaluated using Bio-Revo imaging and analysis software (Keyence, Japan).