Ottix sharper solvents

The site and size of the areas of interest that were analyzed were chosen on the basis of motoric properties of the adult rat body, the knee joint in particular, and are shown in Supplementary Materials, Figure S1. These selected sites are most laden by the weight of the rat’s body. After fixation for 48 h, bones were dehydrated through an ascending ethanol series (30–70%, v/v), fixed with nonpolar Ottix Plus and Ottix Sharper solvents (DiaPath, Martinengo, Italy) and embedded in paraffin blocks. Four-μm-thick sagittal sections of the middle part of the lateral condyle, separated by 10 μm between each section, were obtained using a microtome (HM360, Microm, Walldorf, Germany) [65 (link)]. The cuts were always carried out in the same orientation and plane, and the same regions are presented in all the pictures. The sections were stained with Goldner’s trichrome and then photographed in brightfield light, using a CX43 microscope (Olympus, Tokyo, Japan), equipped with a UC50 digital camera (Olympus, Tokyo, Japan) to evaluate basal morphology of trabeculae. For all analyses, three slides/bone with 20 μm separation were analyzed. The images were analyzed using ImageJ and CellSens (Olympus, Tokyo, Japan) image analysis software [66 (link)].

Two, 10 mm long segments of the small intestine, from the duodenum (2 cm distal to the pylorus) and 50% of the total intestinal length were collected from each bird immediately after slaughter. The tissues were fixed in phosphate-buffered, 4% paraformaldehyde (pH 7.0), dehydrated through a graded ethanol series, fixed with nonpolar Ottix Plus and Ottix Sharper solvents (DiaPath, Martinengo, Italy) and embedded in paraffin. Twenty cros- sections (with 20 μm interval after every 6 slices) of 5 μm thick were cut with an HM 325 microtome (Thermo Fisher Scientific, Waltham, MA, USA) and stained with Masson’s trichrome [37 ]. Microscopic images were collected using a CX43 microscope (Olympus, Tokyo, Japan). The small intestine structure was examined using Olympus cellSens software (Olympus, Tokyo, Japan) and ImageJ [38 (link)]. The following morphometric variables of the intestine were analyzed: villus thickness and length, crypt width and depth, villus length to crypt depth ratio and small intestine absorptive surface, as described by Kisielinski et al. [39 (link)].

Formaldehyde-fixed samples of tibial bone diaphysis were decalcified in a 10% EDTA solution (pH 7.4). Afterwards, the decalcified samples were dehydrated through a graded ethanol series (50%, 70%, 90%, and 100% ethanol in distilled water), fixed with nonpolar Ottix Plus and Ottix Sharper solvents (DiaPath, Martinengo, Italy) and embedded in paraffin. Paraffin-fixed samples were cut with an HM 325 microtome (Thermo Fisher Scientific, Waltham, MA, USA) at a thickness of 5 μm and stained with Picrosirius red (PSR) stain to observe the distribution of mature, organized (seen as red/orange), and immature (seen as green) collagen fibers [55 (link)]. Stained slides were observed in cross-polarized light using a CX43 (Olympus, Tokyo, Japan) microscope.