Ix71 microscope

Cells were fixed in fresh paraformaldehyde solution, and then incubated with PKM2 antibody (1:100, CST) and Cytokeratin antibody (1:100, CST) over night at 4°C and second antibodies were then loaded for 2 h at room temperature in the dark. After washing, Hoechst was used for nuclear staining. Stained cells were visualized with an Olympus Microscope IX71 (Olympus, Tokyo, Japan). Tissues from six mice per group were used for immunofluorescence staining.
Freshly isolated mouse skin specimens were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Nonspecific binding was blocked by treatment with normal goat serum and 0.3% Triton X-100. The tissues were next incubated for 24 h at 4°C with primary rat monoclonal antibodies against PKM2 (1:100, CST) and mouse polyclonal antibodies against Keratin 14 (1:100, Servicebio, Wuhan, China) diluted in primary antibody dilution buffer containing 0.3% Triton X-100. After washing with PBS, immunoreactivity was detected by incubation with FITC with goat anti-rabbit IgG (Servicebio, Wuhan, China) and Cy3 with goat anti-rat IgG (Servicebio, Wuhan, China) diluted in PBS containing 0.5% Triton X-100 for 2 h. An Olympus Microscope IX71 (Olympus, Tokyo, Japan) was used to examine the specimens.

Confluent CellBind Culture Dishes (Corning, NY, USA) were rinsed 2 times with DMEM before adding serum free media and detaching the cells using a cell scraper. Detached cells were pipetted ten times with a 1 mL pipet tip. Using a 10 X objective from a IX71 Olympus microscope, 25–30 fields per dish were captured for analysis of cell aggregates using Image J Software.

The cells were rinsed with PBS twice in the plates after discarding the supernatant and fixed with 4% paraformaldehyde at room temperature for 20 min. The cells were then washed with PBS and stained with oil red O (Solarbio) for 20 min followed by washing with PBS. Lipid vesicles were observed and photographed using an IX71 Olympus microscope (Olympus, Tokyo, Japan). Quantification of the staining was performed using Image-Pro Plus software.

Histological examinations were performed as described previously [23 (link)]; tissues from the fish were dissected, fixed in neutral buffered formalin for 24 h, washed with Dulbecco’s phosphate-buffered saline (DPBS, Sigma, New York, NY, USA), dehydrated with 30% sucrose/DPBS, and embedded in optimum cutting temperature compound (OCT). Eight micrometer sections of all tissue specimens were made using a cryostat (CM1950, Leica, Nussloch, Germany). Sections were stained with hematoxylin and eosin (Sigma, New York, NY, USA) and visualized using a IX71 Olympus microscope coupled to a DP70 Olympus digital camera (Olympus, Tokyo, Japan). Ultrastructural analysis was carried out as previously described [6 (link)]. Transmission electron microscopy (TEM) was performed using a transmission electron microscope (FEI Tecnai Spirit).

Cells were grown to confluency on CellBind Culture Dishes (Corning, NY, USA). Cell monolayers were rinsed twice with DMEM and then cells were detached in DMEM using a cell scraper. Detached cells were dissociated by pipetting ten times with a 1 mL pipet tip. Images were obtained of 25–30 fields per dish using a 10× objective from a IX71 Olympus microscope. Areas of cell clusters with more than 10 cells were measured using Image J Software.