Thunder dmi8

After anaesthetising the mice, the thoracic cavity was opened to expose the heart, and the mice were perfused with cold 0.9% saline from the left heart, followed immediately by 4% paraformaldehyde. Spinal cord sections were removed and post-fixed overnight in 4% paraformaldehyde at 4 ℃. They were then dehydrated in a sucrose gradient solution of 20% and 30% and cut into 10 μm thick cryosections. For immunofluorescence staining, tissue sections were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 15 min, blocked with 5% bovine serum albumin (BSA) for 1 h, and incubated with primary antibodies overnight at 4 ℃. After thorough washing with PBS, cell samples or tissue sections were co-incubated with secondary antibodies (1:200, Jackson ImmunoResearch, USA) for 2 h at room temperature. Sections were counterstained with DAPI and photographed using a Thunder Imager (THUNDER DMi8, LEICA, Germany). Fluorescence quantification was performed using ImageJ software.

BRCA cells were plated into 96‐well plates at a density of 20 000 cells. After 24 h, the culture medium was removed and fresh culture medium containing 100 nM of 25(OH)D or RNA‐Lipo3000 mixture was added. After 96 h after the cells were plated, the EDU assay was performed following the guidelines provided by the manufacturer. The fluorescences of EDU and 4′,6‐diamidino‐2‐phenylindole (DAPI) were collected using THUNDER DMi8 (LECIA). The cell count was determined using ImageJ.

The opening of MPTP is a critical event leading to cell death, which was assessed using an MPTP kit (Beyotime Biotech, Nantong, China) following the manufacturer’s protocol [29 (link)]. Briefly, cells on the coverslip were washed and labeled for 30 min in a dark room at 37 degrees and examined under a Leica Thunder DMi8 microscope. Diminished green fluorescence indicates increased MPTP opening.

Animals were perfused with PBS under deep anesthesia. For each animal, at least two samples from distinct lobules of the liver were collected and fixed immediately in 10% formaldehyde for 24 h. Samples were dehydrated and embedded in paraffin. 6 µm sections were cut from each tissue block and stained with hematoxylin and eosin (H&E). Images were captured using a Leica Thunder DMi8 microscope system at two magnification levels. Lymphocyte infiltration and spotty or widespread necrosis with architectural disturbance or collapse in any area of the liver samples was defined as liver damage.

Cell suspensions (2 × 10⁴ cells) were dropped onto a microscope slide (cat. no. 02286‐AB, SPI Supplies, West Chester, PA, USA), and the slide was incubated at 37 °C for ≈30 min until the cells formed films and attached to the slides. The cells were fixed with 4% paraformaldehyde and blocked with 0.5% TritonTMX‐100. Cells were stained with primary antibodies at 4 °C overnight, followed by incubation with fluorescently labeled secondary antibodies for 1 h at room temperature, protected from light. An anti‐fade solution containing 2‐(4‐amidinophenyl)−6‐indolecarbamidine dihydrochloride (DAPI) was added dropwise, and the coverslips were mounted. The cells were observed and photographed using a Stellaris STED laser confocal microscope (Stellaris STED, Leica, Germany) or a Thunder Imager Fast High‐Resolution Inverted Fluorescence Imaging System (Thunder DMi8, Leica, Germany).

Cells were seeded on cell climbing sheets, fixed with 4% paraformaldehyde. After blocking with QuickBlock™ Blocking Buffer (P0260, Beyotime), cells were given primary antibodies to incubate, including RCAN1 (1:200, D6694, Sigma-Aldrich), YY1 (1:200, ab109228, Abcam), and NFATc1 (1:100, 66963-1-Ig, Proteintech). On the second day, Alexa Fluor 488 goat anti-mouse IgG (H + L) or Alexa Fluor 594 goat anti-rabbit IgG (H + L) secondary antibodies were employed to incubate cells, followed by nuclear staining (0100-20, SouthernBiotech) for 15 min. The fluorescence signals were analyzed by the thunder imager fast high-resolution inverted fluorescence imaging system (THUNDER DMi8, Leica, German).