2030 microtome

Colon samples were removed, fixed in neutral buffered 4% PFA, dehydrated (Shandon Hypercenter, XP), and subsequently embedded in paraffin (TES, Medite). Sections (2–3 μm thick, Reichert-Jung 2030 microtome) were deparaffinized in xylene and H&E stained according to standard protocols. Histological scoring was performed in a blinded manner as described previously.^{23 (link)}

The testes with attached epididymides and livers were fixed in Bouin's fluid and processed for paraffin wax embedding according to routine protocols. Sections of 7 μm in thickness were obtained with Reichert-Jung 2030 microtome. Sections were stained with haemalum/eosin to show general morphology, with Mallory's trichrome modified by Galgano [27 ], to view connective tissue fibers, or used for immunohistochemistry (IHC). All the histological results were examined by using a Nikon-MicroPhot-FXA light microscope.

Formalin-fixed hind legs were decalcified for 14 days in 18% EDTA and frontally embedded in paraffin blocks. Blocks were cut using a Reichert-Jung 2030 microtome to produce 5μm thick sections. Sections were deparaffinized then stained with Weigert’s iron hematoxylin/fast green/safranin-O (Sigma). To systematically compare knee articular cartilages, a minimum of five equally spaced sections spanning the knee joint were analyzed using a modified literature semi-quantitative scoring criteria [23 (link)]. Articular cartilage with loss of staining or superficial structural damage was referred to as mild OA (scores of 0.5 and 1), while fibrillations and erosions with increasing depth and width below the superficial layer and extending past tidemark were referred to as moderate (scores of 2 and 3) or severe OA (score of 4). Sections with majority of the subchondral bone exposed or only a small portion of articular cartilage intact were categorized as very severe OA (scores of 5 and 6). The medial femoral and tibial articular cartilages of each knee section were blindly scored by 7 trained examiners. The scores from each examiner were averaged and the average scores for the femoral and tibial cartilage were plotted separately on the same graph for semi-quantitative comparisons.