Quantimet

Cardiac chambers were then fixed by immersion in 4% buffered formalin and embedded in paraffin for histological processing. Sections (4 µm) were stained with hematoxylin and eosin for examination by light microscopy. Only nucleated cardiac myocyte from areas of transversely cut muscle fibers were included in the analysis [6 (link)]. Quantification of left ventricular fibrosis was achieved by picrosirius red staining. Analyses were performed in a computer-assisted morphometric system (Leica Quantimet, Cambridge, UK).

For analysis of CSA, the tibialis anterior muscle was chosen as it is composed mainly of glycolytic fibres, which are the most affected in CKD 22, 23. The muscles were removed and frozen in liquid nitrogen. The samples were cut transversally in 6‐μm sections in a cryostat (LEICA CM 1100) and stained with haematoxylin and eosin for examination by light microscopy. Image capture and analysis were performed in a computer‐assisted morphometric system (Leica Quantimet, Cambridge, United Kingdom) with 200× magnification and 20x objective. Images were further analysed on a digitalizing unit connected to a computer using the Quantimet program (Leica Qwim). A minimum of 300 fibres in five different fields were analysed for each animal. All analyses were conducted by a single observer (EM), blinded to the rat's identity.