Tissue processor

Skin tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 48 h at 4 °C. The fixed tissues were dehydrated in increasing concentrations of ethanol, cleared in xylene (Duksan, Seoul, Republic of Korea), and embedded in paraffin using a tissue processor (Leica, Wetzlar, Germany). The paraffin-embedded tissue blocks were sectioned into 7 μm slices using a microtome (Thermo Fisher Scientific, Rockford, IL, USA). The sections were mounted on coated microscope slides (Muto pure chemical Co., Ltd., Tokyo, Japan) and baked at 60 °C for 24 h to enhance tissue adhesion.

Skin tissue was fixed in cold 4% paraformaldehyde (Sigma-Aldrich) for 48 h, placed in a cassette, and washed with DW. Then, the sample was placed in a tissue processor (Leica, Wetzlar, Germany), sequentially soaked in 95% and 99% ethanol (Duksan), dehydrated, dipped in xylene (Duksan), and infiltrated with paraffin (Leica). Tissue blocks soaked in paraffin were made into paraffin blocks in an embedding machine, sectioned to a thickness of 7 µm using a microtome (Leica), placed on a coated slide, incubated overnight at 60 °C, and attached to the slide.

Following 24 h of fixation in 4% formaldehyde, the brain tissues were processed using a Leica Tissue Processor according to a standard protocol for mouse tissues. In summary, the tissues were dehydrated in a gradient of ethanol (70%, 95% and 100%), cleared in xylene, infiltrated with paraffin wax, and embedded into paraffin blocks. These blocks were then sliced at a thickness of 5 µm using a microtome (Leica, Wetzlar, Germany). The sections were stained with H&E and examined using an Axio Observer Inverted Microscopy (Zeiss, Munich, Germany).
For each section, four microscopic fields at 40× magnification were captured in the cortex, caudoputamen (Cpu), and hippocampus and subsequently quantified. Cells exhibiting bright red eosinophilic staining with condensed triangular nuclei were identified and counted as damaged neurons.

Operated knees were collected, fixed in 10% neutral buffered formalin for 48 h, and then stored in 70% ethanol before proceeding to decalcification. Then, the knees were decalcified in 10% formic acid for 2 weeks and infiltrated with paraffin wax via a Leica Tissue Processor at the UF Molecular Pathology Core Facility. After embedding in paraffin wax, the joints were sectioned in the frontal plane (10 μm), with sections representing the loading region of the joint being stained with safranin-o and fast green or hematoxylin and eosin. Using an open-source program developed in-house [20 (link)], user-defined regions of interest were used to calculate the minimum cartilage thickness, osteophyte area, and maximum subchondral trabecular bone area to total subchondral area (bone + marrow) in the tibial region of the medial joint compartment.
In addition to these quantitative methods, semi-quantitative scores were assigned to each joint. These scores included OARSI grades [21 (link)] or both medial and lateral compartments of the joint and synovitis score of the medial compartment [22 (link)]. Grades were assigned based on the consensus of four scorers blinded with respect to the group.

UPS tumors were fixed in 10% neutral buffered formalin (Research Products International Corp) for 24 hours and embedded into paraffin using a tissue processor (Leica). The tissues were sectioned to 5 microns (Leica RM2255) and stained with hematoxylin and eosin (H&E) following the same protocol as Foothills Medical Centre Calgary Laboratory Services.

Fixed lung tissues were paraffinized using a tissue processor (Leica, Wetzlar, Germany, 0422). The tissues then were sectioned at 5 μm using a microtome (Leica, Wetzlar, Germany, 9224) and were stained using an H&E staining kit (Abcam, Dawinbio Inc., Hanam, Gyeonggi-do, Republic of Korea, ab245880). The stained samples were examined under an optical microscope (Nikon, Tokyo, Japan, Eclipse 80i), and the images were graded from 0 to 4. The grades used were: 0, no injury (normal); 1, minimal (injury up to 25% of the field); 2, mild (injury between 25 and 50% of the field); 3, moderate (injury between 50 and 75% of the field); and 4, severe (injury over 75% of the field), for thickening of the alveolar-capillary walls, the numbers of infiltrated cells, hemorrhage, and the vascular congestion.

At the end of the experiment, fresh liver samples from each group were placed in a tissue embedding cassette and fixed in 10% neutral-buffered formalin solution at 4°C overnight. The next day, samples were transferred to 70% ethanol and processed with a tissue processor (Leica, Wetzlar, Germany). The processed liver samples were then embedded in paraffin and cut into 5 µm thickness sections followed by hematoxylin and eosin (H&E) staining for microscopic examination. Images (five from each section) were captured randomly at 20x magnification and scored in a double-blind manner. Hepatocyte ballooning was quantified as described by Kleiner et al. (Kleiner et al., 2005 (link)) and the average score of all the samples from the same group was reported.