Total 5 random 4×10 magnification fields were selected and average number of colonies of each sample was calculated.
The observations coming from three magnification fields were averaged.
Four randomly selected × 200 magnification fields were photographed, and the number of invading cells was counted.
Ten high magnification fields were observed for each slice and 100 cancer cells were counted in each field.
Five high-power (200x magnification) fields were chosen randomly for each section, and 500 cells were counted per field.
Three high magnification fields were counted for each tumour section and the mean microvessel density value was recorded for each.
For the enumeration of cell-in-cell frequency per sample, cells from 40× magnification fields were counted and the cell-in-cell frequency was represented as mean ± SEM from at least three different fields.
The stained slides were viewed with a light microscope (Nikon Eclipse 80i), and digital images of six randomly selected, high-power (×400 magnification) fields were captured on NIS- ElementS.F.2.30 software (Nikon United Kingdom Ltd., Surrey, UK).
For calculation of HoCC frequency (percentage of cannibal cells per sample), cells from three 20× magnification fields were counted per sample, and cannibalism was expressed as the mean ± SEM of three different samples per cohort.
Cortical staining intensity was scored on a scale of 0 to 3 (0 no staining, 1 mild, 2 moderate, 3 intense) for E-cadherin (distal tubules), α-SMA (interstitium), MnSOD (tubules), Nox-2 (interstitium), p-smad2 and superoxide (tubulointerstitium). Five high magnification fields were evaluated per kidney and results were expressed as mean and standard error bars.
