Paraformaldehyde (pfa)

EPC cells were grown on glass coverslips in 24-well plates overnight, fixed with 4% PFA (Beyotime). After being washed three times with PBS and treated with 0.1% Triton X-100 (Sigma) to permeabilize for 15 min. After three washes in PBS, EPC cells were stained with 1 μg/ml DAPI (Beyotime) for 15 min in the dark at room temperature. Finally, the coverslips were washed and observed with a confocal microscope under a ×63 oil immersion objective (SP8; Leica).

HeLa and HeLa ICP47 cells were seeded onto the glass coverslips in 24-well plates (0.6 X 10⁵ cells/well) before transfection. The transfected cells were washed with PBS and fixed with 4% PFA (Beyotime, Shanghai, China) for 30 min. The samples were blocked in 3% BSA for 30 min, then stained with Acti-stain™555 (Cat. PHDH1, Cytoskeleton, CO, USA) and DAPI (Beyotime, Shanghai, China) for 30 min. Samples were mounted with mowiol (Cat. 10852, Sigma-Aldrich, MO, USA) on microscope slides and imaged using Leica SP8 confocal microscope (Leica, Nussloch, Germany). Images were recorded for at least 50 cells per HLA I allotype in three independent experiments. Images were analyzed by ImageJ software (version 1.53, National Institutes of Health, MD, USA).

Histological analysis was conducted to investigate the effects of cold exposure on tissue structure. The fish were exposed to cold stress as described above. After cold exposure, the fish were euthanized by immersion into ice-slurry for 3 min as previously described [21 (link)]. The fish were dissected and tissues including brain (cerebellum), gill, heart, kidney, liver, muscle and spleen were collected. The samples were fixed in 4% PFA (Beyotime Biotechnology, Shanghai, China) at 4 °C overnight. After fixation, the samples were dehydrated with alcohol gradients, embedded with paraffin and sectioned into 4 μm slices. The slices were stuck to glass slides, soaked in xylene for dewaxing and rehydrated in ethanol gradients. Finally, the slides were subjected to H&E staining. Photographs of the tissue sections were taken using an Aperio VERSA Brightfield, Fluorescence & FISH Digital Pathology Scanner from Leica (Wetzlar, Germany). Subjective analyses were performed for the photographs to identify changes of tissue structure among the samples from different experimental groups. The photographs were examined independently by different investigators to avoid personal preconceptions when making their judgments.

The subcellular localization of circLMO1 was evaluated using FISH assay. Specific RNA probes against cricMLO1 and miR-4291 was synthesized using FISH Tag™ RNA Kit (F32952; Invitrogen). CaSki and C33A cells (about 1.5×10⁴/well) were mounted on a coverslip and fixed with 4% PFA (Beyotime) at room temperature for 15 min. The cells were digested with protein K at 37°C for 1 h in the presence of glycine and acetic anhydride. Then, cells were treated with pre-hybridization solution for 90 min and treated with the probe (300 μL, 250 ng/mL) against cricMLO1 at 42°C overnight. Finally, cells were stained with DAPI for 5 min at room temperature before sealing. A fluorescence microscope (Keyence, Osaka, Japan) was used to capture the signals of cricMLO1, miR-4291 and the nucleus.

Migration and invasion were examined utilizing Transwell chambers (Corning, Shanghai, China). For invasion test, the chambers were coated by Matrigel (BD, USA), without Matrigel for migration test. HCC cells were inoculated onto the upper chambers plus serum-free media (3 × 10⁴ cells/well). DMEM media with 10% FBS were added to the lower chambers. Following 24 h, migrated or invasive cells were fixed by 4% PFA (Beyotime, China), and dyed utilizing crystal violet. The number of migrated or invaded cells was counted at ×100 magnification utilizing an inverted light microscope.

Proliferation of the cells was examined using a BeyoClick™ EdU-647 kit (Beyotime). In short, the cells were sorted in 6-well plates. After adherence, the medium was renewed, and each well was loaded with 10 μM EdU solution and the cells were incubated at 37℃ for 2.5 h. Next, the cells were fixed in 4% PFA (Beyotime) for 15 min, permeabilized in 0.3% Triton X-100 (Elabscience Biotechnology Co., Ltd., Wuhan, Hubei, China) for 8 min, and then added with 500 μL Apollo staining buffer in the dark for 40 min. The nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) for 10 min. The staining was observed under a fluorescence microscope (Zeiss, Oberkochen, Germany) and quantified using the Image J software.

Chondrocytes were seeded into a 12-well plate. After the cells were grown to 80% concentration, the medium was removed and washed 2 times with PBS. 4% PFA (Beyotime) fixed cells for 10 min, then PBS washed 3 times. Alcian blue staining solution (ALCB-10001, OriCell, Guangzhou, China) was added to the wells and stained for 60 min (37 °C). Remove the staining solution, wash 3 times with distilled water, and observe under the microscope.