Masson’s trichrome staining.

Kidney paraffin sections (4–5 µm thick) were stained with Masson’s trichrome to evaluate the severity of renal fibrosis. The staining reagents were purchased from Sigma, and staining was performed according to the protocol provided by the manufacturer. However, in one kidney sample from a male and female eNOS^−/− db/db mouse, we used made-in-house reagents to stain the sections by following the protocols previously described (4). Images were captured using an Olympus DP70 digital camera with DP manager software (version 2.2.1). Fibrotic areas (stained blue by Masson’s trichrome) were quantified as a total number of pixels by a blind observer using the neural networks machine learning algorithm in the R environment. The neural network model used to count the fibrotic area was built using the “neuralnet” package in the R environment. The model has an accuracy rate of 96% on randomly selected samples.