Histological Evaluation of Tissue Inflammation

Heart and skeletal muscle tissue sections were fixed by incubation in 10% buffered formalin for 24 h and dehydrated in absolute alcohol. Tissue samples were then cleared in xylene and embedded in paraffin. Slides containing 5-micron paraffin-embedded tissue sections were stained with hematoxylin (stains nuclei blue) and eosin (stains extracellular matrix and cytoplasm pink). The H&E stained tissue slides were imaged at 20 × magnification by using an Olympus BX-15 microscope (Center Valley, PA) equipped with digital camera and Simple PCI software (v.6.0, Compix, Sewickley, PA). The inflammatory infiltrate and tissue damage were scored as described previously (22 (link)). Briefly, scoring was defined as (0) - absent/none, (1) - focal or mild with ≤1 foci, (2) - moderate with ≥2 inflammatory foci, (3) - extensive with generalized coalescing of inflammatory foci or disseminated inflammation, (4) - severe with diffused inflammation, interstitial edema, and loss of tissue integrity (22 (link)). For each group, data were captured from at least three mice, two slides per tissue, and ten microscopic fields per slide, and presented as mean value ± standard error mean (SEM).