Neg 50

Zebrafish at 3 dpf, 5 dpf, 4.9 mm SL, 5.9 mm SL, 6.4 mm SL, 7.2 mm SL, 8.6 mm SL, 11 mm SL, and 13 mm SL were collected, anesthetized (see above), and killed by cutting their heads. Up to 13 mm SL, heads were directly fixed in 4% PFA. For adults, eyes were dissected. One eye was used for RNA extraction and the other was fixed in 4% PFA overnight at 4°C. Fixed heads and eyes were washed twice for 10 min in 1X PBS and kept for 1 hr in 5% sucrose in 1X PBS, followed by overnight incubation in 30% Sucrose in 1X PBS at 4°C. Finally, eyes were incubated for 1 hr at room temperature in a 1:1 solution of 30% Sucrose in 1X PBS/ NEG‐50™ (Thermo Fisher Scientific) and mounted in NEG‐50™ and frozen in dry ice. All samples were kept at ‐80°C until sectioning. Zebrafish eyes were cut into 16‐μm sections and left to dry for at least 1 hr at room temperature. After sectioning, all samples were kept at ‐20°C for a maximum of 3 weeks. Before staining and imaging, retinal sections were dried for at least 1 hr at room temperature followed by rehydration in 1X PBS for 30 min. For nuclear staining, sections were incubated for 1 min in DAPI (Thermo Fischer Scientific) diluted 1:10000 in PBS at room temperature. Finally, sections were washed three times for 5 min at room temperature and mounted in Vectashield antifade mounting medium (Vector Labs).

Liver samples were fixed using 4% (v/v) paraformaldehyde overnight at 4°C. The next day, samples were cryopreserved using sterile-filtered 30% sucrose (w/v) in dulbecco’s phosphate buffered saline (DPBS) (Gibco, 14190-144). After 3 days, each sample was placed in a 1:1 30% sucrose:Neg-50 (Richard-Allan Scientific) solution and incubated overnight at 4°C. The samples were frozen on dry ice using undiluted Neg-50 at −50°C and stored at −80°C until sectioning. Sequential 20 μM thick sections were obtained from each sample using a Leica CM3050S cryostat.
For nuclei and LD labeling, sectioned tissue was washed three times at room temperature in DPBS for 5 min each. Afterward, DPBS containing 10 μM BODIPY (Invitrogen, #D3922) was placed on the samples and incubated for 30 min at room temperature in the dark. Next, the slides were washed with DPBS twice before incubating in DPBS containing 5 μg/ml DAPI (Invitrogen, D1306) for 10 min at room temperature in the dark. After DAPI staining, the slides were washed three times in DPBS for 5 min each before being mounted using SlowFade Diamond antifade (Invitrogen, #S36972) and sealing with nail polish overnight. Slides were imaged immediately using a Zeiss LSM710 confocal microscope. Images were developed using the IMARIS (Bitplane) image analysis software suite.

At day 90 after surgery, menisci were harvested and placed in a pre-labeled base mold filled with frozen section medium (Neg 50; Richard-Allan Scientific; Kalamazoo, MI). The base mold with tissue samples was quickly immersed in liquid nitrogen cold 2-methylbutane and allowed to solidify completely. The tissue blocks were then either placed on dry ice and subsequently stored in a deep freezer (− 80 °C) until used for histological analysis or cut into 10-μm-thick sections, which were fixed with 4% paraformaldehyde for 30 min. The fixed tissue sections were stained with either hematoxylin and eosin (H&E) or safranin O and fast green according to standard protocols.

Following either sham or MCAO surgery, mice were anaesthetized with pentobarbital (100 mg/kg, i.p.) and transcardiacally perfused with ice-cold ACSF. Brains were instantly extracted and embedded in NEG50 (Richard-Allan Scientific, Fisher Scientific, Switzerland) and stored at −80°C. For immunostaining, 30 μm sections were cut using a cryostat (Hyrax 60, Carl Zeiss) and mounted onto glass slides (SuperFrost Plus, Thermo Scientific). The sections were fixated using 4% PFA for 30 min at room temperature, followed by antigen retrieval in a PickCell 2100-Retriever in 10 mM citrate buffer pH 6.0, 0.05% Tween 20 for 20 min. Sections were then permeabilized (0.2% Triton X-100 in 10 mM Tris pH 7.4, 150 mM NaCl, 10% NGS) and incubated with primary antibody diluted in TBST (10 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween-20) containing 10% NGS overnight at 4°C. After washing five times for 5 min with TBST, the sections were incubated with secondary antibodies (Alexa Fluor Plus 800) for 1 h at room temperature. After washing with TBST, sections were air dried in the dark and scanned with an Odyssey CLx infrared scanner (Licor) at a resolution of 21 μm (Figures 4A, 6A) and 42 mm (Figure 3C).

To verify the effect of PGE₂ treatment on the differentiation of hTSCs in vivo, eight female nude rats (10 weeks old; 200–250 g) were used. hTSCs at passage 2 were seeded into 24-well plates (8×10⁶ cells/well) and cultured in DMEM with or without various concentrations of PGE₂ for 6 days, with a change of medium every day. For implantation experiments, the cells were trypsinized from each well and mixed with 0.25 ml Matrigel (BD Scientific) to enable gel formation after implantation. These hTSC-Matrigel mixtures were placed subcutaneously in the back of anesthetized rats. Three pieces of hTSC-Matrigel were positioned in three distinct places on each side of each rat’s back. Four weeks after implantation, tissue samples were harvested from the implanted area and placed in pre-labeled base molds filled with frozen section medium (Neg 50; Richard-Allan Scientific; Kalamazoo, MI). The tissue blocks were stored at −80°C until histological analysis.

The effect of MTR on aging rat patellar tendons was determined by histological analysis according to previous descriptions [22 (link)]. Briefly, patellar tendons were dissected from 3 rats in each of the control and MTR groups and immersed in frozen section medium (Neg 50; Richard-Allan Scientific; Kalamazoo, MI). Then, they were flash frozen in 2-methylbutane chilled in liquid nitrogen, sectioned (10 μm), dried overnight at room temperature, rinsed in PBS, and fixed in 4% paraformaldehyde for 30 min. Finally, the sections were washed in PBS, stained with H & E and visualized through a microscope.