Rm2135 microtome

Two, 4, 8, and 12 weeks after surgery, tibial specimens (3 mm from both distal and proximal ends of the normal tibia) were taken from the surgery side of four rabbits in each group, and fixed in 10% formalin (Yilin Chemical Plant, Nanjing, China). The specimens were decalcified, soaked in gelatin, embedded in methyl methacrylate (Yilin Chemical Plant, Nanjing, China), and serially sectioned into 5-μm slices using a freezing microtome (RM 2135 Microtome, Leica, Germany); the slices were stained with hematoxylin and eosin (automated tissue stainer, ZMN-2802) and examined with ×10 optical microscopy (Olympus, Tokyo, Japan) for histological changes during the bone graft repairing process. The images of slices were analyzed to calculate the new bone quantity using Mias Sharpvision image analytical system. When the specimen section was visualized clearly in the field, the image scale was calibrated, and the total bone tissue areas, including the surrounding new bone callus and the new bone callus area only, were measured in slices from different time points in both groups. The formula for calculation of new bone quantity was as follows: new bone quantity (%) = new bone area (mm²) / total sectional area of bone (mm²) × 100%.

After fixation in 4% PFA as described above, membranes were cut from their supports with a scalpel and dehydrated through a series of baths in increasing concentrations of ethanol in water (50%, 75%, 95%, 100%, 100%). After clearing 10 min in Clearene (Leica, Germany) to replace the ethanol, samples were immersed in liquid paraffin for one hour at 60 degrees to allow complete infusion of paraffin into the cells, and then set into block moulds and allowed to solidify at RT. Sectioning was performed on a Leica RM2135 microtome, and cut sections were deposited onto slides with the aid of a heated water bath. Prior to staining or immunolabelling, paraffin was removed with 2x10 min washes in Clearene, and then immediately rehydrated with the same series of ethanol baths in reverse order. For epitope retrieval, slides were immersed in citrate buffer at 90°C for 20min.

The ovary and uterine tissues were fixed in 10% buffered formalin and then dehydrated and embedded in paraffin. The paraffinized tissue blocks were cut into 4 μm thick sections using a Leica RM2135 Microtome (Leica, Germany). After assembly into a glass slide, the tissue was deparaffinized in xylene, rehydrated in graded ethanol, and submitted to hematoxylin and eosin (H&E) staining and sealing with Entellan® (Merck Millipore, Germany). The H&E slides were then reviewed and examined using a light microscope (Primostar, Zeiss, US).

The skin samples were dehydrated with EthOH in successive concentrations of 70, 80, 90, 95, 99, and 100%. The samples were further infiltrated three times in xylene. Each dehydration step was performed in a dehydration system (Leica TM 1020, Germany) for 22 h, and the tissues were then paraffin-embedded. We investigated skin anatomy in cross-sections and vertical sections. The skin tissue samples were cut using microtome blades on a Leica RM 2135 microtome. The sections were stained with L-Dopa (Solarbio, China). Images of the tissue sections were obtained on an Olympus SZX16 stereomicroscope (Japan). Finally, the melanin content of the barb ridges was analyzed using IPP 6.0 (Image-Pro Plus 6.0). The melanin content was expressed by calculating the area occupied by the melanin, which was stained dopa in the cross-section of the feather follicles.

The lung tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Four-micrometer-thick sections were cut with a Leica RM2135 Microtome (Germany). After xylene deparaffination, rehydration, and antigen retrieval, the sections were blocked in 10% donkey serum blocking buffer. Slides were then incubated in primary antibodies overnight at 4°C, followed by being incubated with HRP-conjugated secondary antibody for 1 h at RT. The antigen–antibody binding was detected with DAB substrate solution. The sections were counterstained with hematoxylin for nuclei. The staining was observed and imaged under the microscope (BA400Digital, Motic, USA). Immunohistochemical (IHC) images were analyzed using ImageJ-IHC profiler for semi-quantification as described elsewhere (Crowe and Yue, 2019 (link)). GraphPad 8.0.1 was used for statistical analysis.