Rm2245

Nissl staining was used to observe the number and morphology of neurons. Brain tissue was prepared as above and coronal sections were cut with a thickness of 5 μm on a paraffin microtome (RM2245, Leica, Bensheim, Hesse, Germany) after paraffin embedding. Brain sections were then deparaffinized, rehydrated, stained with cresyl violet, mounted with neutral balata, and covered with coverslips. Finally, the sections were examined under a microscope (DM 4000B, Leica, Wetzlar, Hesse-Darmstadt, Germany).

Hematoxylin-eosin staining (HE): Left ventricular tissue was placed in 4% polyformaldehyde, embedded in paraffin and cut into 4-5μm thick serial sections. Slices were deparaffinized in a sectioning machine (LEICA RM 2245), treated with xylene, stained with hematoxylin-eosin and dehydrated by subsequent immersions in graded alcohol solutions, and were then sealed with neutral gum. Myocardial structure was visualized using an optical microscope (Olympus, Tokyo, Japan, BX53).
Transmission Electron Microscopy (TEM): The cardiac left ventricle was fixed in 3% glutaraldehyde, subjected to 1% tetroxide re-fixation, pyruvate dehydration, embedded in epoxy resin (Epon) 812, and cut into 50nm thin slices. Uranium acetate and lead citrate double staining was performed and tissue slices were viewed with a H-600IV transmission electron microscope.

For miRNAs extraction, eight sections of 20 μm each were cut from FFPE eyes on a RM2245 microtome (Leica, Bannockburn, IL, USA). Sections were transferred to glass slides, and tumor tissue was isolated by hand with a scalpel. We also analyzed FFPE from five unaffected eyes. RNAs were extracted by using Recover All Total Nucleic Acid Isolation Kit (Ambion), following the manufacturer's protocol. The expression of miRNAs from FFPE samples was analyzed by TaqMan MicroRNA Assay, as previously specified.

On the last day of the wound healing experiment, the animals were anesthetized using Ketamine HCl (50 mg/kg, i.p.), euthanized, and specimens of wound tissue with the adjacent healthy tissue were collected. The collected samples were fixed in 10% formalin and were subjected to routine histopathological tissue examination. The wound tissue specimen was sectioned with a microtome (Leica RM 2245) and then stained with hematoxylin-eosin. The prepared tissue slide was examined under a light microscope. To evaluate the collagen content, the wound tissue specimen was sectioned using a microtome, stained with Van Gieson stain for collagen fiber, and examined under a microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany).

Mice were anesthetized with isofluorane and perfused with PBS followed by 4% paraformaldehyde (PFA). After postfixation with 4% PFA overnight, the brains were dehydrated using a graded series of alcohol, cleared in xylene, and embedded in paraffin. Then, coronal slices (40 μm) were cut by a rotary microtome (LEICA, RM2245, Heidelberg, Germany) for the hypothalamic paraventricular nucleus (PVN). After deparaffinization, sections of the brain were incubated in 3% H₂O₂ for 15 min to block endogenous peroxidases for antigen retrieval and then washed with phosphate-buffered solution (PBS). Next, sections were incubated with 5% goat serum and 0.01% Triton-100 (dissolved in PBS) for 1 h and then stained with primary antibodies (PSD95, 1:400, abcam, Cat# ab12093, Cambridge, UK; FKBP51, 1:200, Proteintech, Chicago, IL, USA) overnight at 4 °C. Following washing with PBS, the sections were incubated with Alexa Fluor 488 or 555 donkey antimouse, antigoat, or antirabbit secondary antibodies (1:1000 dilution, Invitrogen Life Technologies, NY, USA). Images were captured by a fluorescence microscope (Zeiss, Heidelberg, Germany) after incubation with 4-6-diamidino-2-phenylindole (DAPI) for 15 min and washing with PBS.

After μCT imaging, harvested samples were prepared for histological analysis. For this, fixed specimens were decalcified with RapidCal^TM solution (BBC Chemical Co., Stanwood, WA, USA), and dehydrated in a graded series of increasing ethanol dilutions (from 70% to 100%). To obtain thin sections, samples were bisected in the middle of the defect and embedded in paraffin. The blocks were serially cut into 5 μm thick sections using a microtome (RM2245, Leica, Germany), and histological preparations were prepared. The sections were placed on slide glass and stained with hematoxylin-eosin (HE) and Masson’s trichrome (MT) stain using standard techniques to check biocompatibility and periodontal tissue regeneration. Under a light microscopy (IX71, Olympus, Tokyo, Japan), digital images were photographed using Meta-Morph, and the results of periodontal tissue regeneration within the defect were presented.

COs were washed two times for 5 min at room temperature in 5 ml of PBS in 5-ml round-bottom tubes (#1152367; BD Falcon) and then fixed overnight in 4% PFA at 4°C on a rocker. The samples were washed three times for 15 min in PBS and then sequentially dehydrated in 40%, 70%, and 95% ethanol diluted in ddH₂O for 1 h each, 95% ethanol overnight, and then three times for 1 h in 100% ethanol at 4°C on a rocker. COs were then soaked in xylene for 5–10 min at room temperature in biopsy cassettes (#M506-3; Simport). The samples were air-dried for 2–3 min and washed twice in melted paraffin (#39602004; Leica) at 60°C for 1 h and once overnight. The samples were embedded into paraffin blocks within 16 h, sectioned using 80-mm microtome blades (MB35; Thermo Fisher Scientific) at 14 or 20 μm on a semiautomated rotary microtome (RM2245; Leica), mounted on SuperFrost Plus Microscope Slides in distilled H₂O heated to 45°C, and then dried overnight at 45°C and stored at 4°C.