Fluoroshield medium with dapi

Immunofluorescence assays were conducted to analyze the translocation of zonulin, where HT-29 cells were seeded on cover slips at 3 × 10⁵ cells/well (24-well plates). After complete confluence had been reached, HT-CECT-7347, and TNF-α (4 ng/mL) were added to HT-29 cells for 16 h. After treatment, cells were fixed with formaldehyde 4% v/v for 10 min at 4 °C and then permeabilized with 0.25% Triton-X100 for 10 min. Next, the cells were blocked with 2% w/v bovine serum albumin in PBST + glycine. The samples were incubated with zonulin (5 μg/mL, Thermo) antibodies at 4 °C overnight, followed by incubating with an AlexaFLuor488-conjugated goat anti-rabbit (1:1000, Abcam) for 2 h at room temperature. Fluoroshield medium with DAPI (Abcam) was used for visualization of nucleus. The stained cells were examined by confocal laser scanning microscope (FV1000, Olympus, in the microscopy section of the SCSIE at the University of Valencia).

The distribution of cells (10 000) in the cell cycle was determined using flow cytometry as described previously [14 (link)]. The fraction of cells expressing p16, p21, and p53 cell cycle inhibitors was quantified using immunofluorescence. Cells were fixed with 4% paraformaldehyde and 100% methanol, permeabilized with 0.1% Triton-X in PBS for 10 min, and blocked with a solution containing 1% bovine serum albumin, 22.5 mg/mL glycine, and 0.1% Triton-X in PBS for 30 min. Afterwards, specimens were incubated with antibodies directed against p16 (#ab108349; Abcam), p21 (#2947, Cell Signaling Technology, Danvers, MA, USA), and p53 (#2527, Cell Signaling), prepared at 1:200 dilution, for 24 h, at 4 °C. Then, the cells were extensively washed and subjected to DyLight 488 IgG (#ab96899, Abcam; 1:500) for 1.5 h at room temperature. After the incubation, the specimens were preserved with Fluoroshield medium with DAPI (Abcam) and inspected under a fluorescence microscope. By moving from left to right and from top to bottom, 500 cells were analyzed.

A different group of mice was used for immunohistochemistry studies. After 9 h of fasting or ad libitum, the mice were anesthetized with 1.4% isoflurane for 3 min and perfused transcardially with PBS followed by 4% cold, buffered paraformaldehyde. The brain tissues were removed and post-fixed at 4°C overnight, and then cryoprotected in 15% sucrose followed by 30% sucrose in PBS for 48 h. 20-μm coronal sections were obtained with a Leica CM1850 cryostat. Non-specific antibody binding was inhibited by incubating the slices in 0.1 M PBS containing 10% goat serum and 0.3% Triton X-100. Slices were then incubated for 24 h at 4°C with one of the following primary antibodies: rabbit anti-c-Fos antibody (#2250, 1:200 dilution, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-caspase-3 antibody (#9662, 1:200 dilution, Cell Signaling Technology, Danvers, MA, United States), and rabbit anti-cleaved caspase-3 antibody (#9661, 1:200 dilution, Cell Signaling Technology, Danvers, MA, United States). After the incubation, tissue sections were washed and incubated for 1 h at room temperature with Alexa Fluor 488 or Alexa Fluor 594 (1:500 dilution, Thermo Fisher Scientific, Rockford, IL, United States) as the secondary antibodies. Fluoroshield medium with DAPI (ab104139, Abcam, Cambridge, MA, United States) was used for nuclear counterstain.