Leica rm2235 microtome

For segmental defect samples, all constructs that were not being processed for vascular-CT imaging, were decalcified in Decalcifying Solution-Lite (Sigma-Aldrich) for 1 week before tissue processing. Once decalcified, all samples were dehydrated and embedded in paraffin using an automatic tissue processor (Leica ASP300, Leica). All samples were sectioned with a thickness of 8 μm using a rotary microtome (Leica Microtome RM2235, Leica). Sections were stained with H&E for vessel infiltration, Safranin O to assess sulphated glycosaminoglycans (sGAG) content, and Goldner’s trichrome for bone formation. Quantitative analysis was performed on multiple H&E-stained slices, whereby vessels (positive staining for endothelium and erythrocytes present within the lumen), were counted on separate sections taken throughout each construct and averaged for each construct. Safranin O sections were evaluated for new developing bone (positive sGAG content). Masson’s trichrome–stained sections were evaluated for new bone formation. The percentage of developing bone, new bone, and marrow per total area of construct was measured in separate sections with the Deconvolution ImageJ plugin.

At the end of the feeding trial, piglets were euthanized after being fasted for 12 h. Uteri were then rapidly isolated from the surrounding fat and tissue under sterile conditions and weighed to calculate the relative weight (relative weight of uteri [g/kg] = uterine weight/live pig weight). Two samples of uterine tissue from each pig were rapidly collected, one of which was collected in an RNase-free 2-mL frozen tube and placed in liquid nitrogen, and then stored at −80°C for subsequent analysis of the relative mRNA expression of GHR. The second sample was promptly fixed in Bouin’s solution for immunohistochemical analysis. Following fixation, 5-μm sections were cut on a Leica RM 2235 microtome (Leica, Germany), mounted on poly-L-lysine-coated glass slides, and dried overnight at 37°C prior to routine staining for immunohistochemical analysis.

For histologic examination, intradermal challenges on the abdomen skin were inducted on Day 40. After 20 min, 24 h and 48 h, the mice were sacrificed using cervical dislocation, and the challenge site was removed and placed in 10% neutral buffered formalin overnight at room temperature. The skin tissue was dehydrated gradually through 70%, 80% and 95% and absolute ethanol (Guo et al., 2020 (link)), then soaked in xylene for 1 h at 55–60°C followed by embedding in paraffin for 1 h. The specimens were sectioned to a thickness of 5 µm using a Leica RM2235 microtome (Leica) and followed by deparaffinisation and haematoxylin and eosin stain. The sections were also stained with rabbit anti‐CD4 (1:200) and rat anti‐CD8 (1:200) monoclonal antibodies (NovusBiologicals) to detect cluster of differentiation (CD)‐maker positive cells: The sections were incubated with peroxidase‐labelled goat anti‐rabbit and goat anti‐rat antibodies and stained in the substrate solution containing 3,3′‐diaminobenzidine (ZSGB‐BIO). Inflammatory cell infiltration was examined using light microscopy, and the images were taken using the microscopic camera system BA400 Digital (Motic Group Co. Ltd.).