Bx43 fluorescence microscope

Transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was executed with a TUNEL staining kit following the manufacturer’s protocol (Roche Inc., Basel, Switzerland). Sections of rat hippocampus were cultured with TUNEL reaction mixture containing TdT enzyme as well as fluorescein isothiocyanate (FITC) labeled dUTP in a dark and humid room for 1 h at 37°C, followed by a final wash for 15 min and visualized using a converter peroxidase (POD) with 4’,6-diamidino-2-phenylindole (DAPI). The number of TUNEL positive cells for five fields in the bilateral hippocampus of each section was counted under an Olympus BX43 fluorescence microscope (Olympus, Tokyo, Japan). An average for the five slices per brain was taken.

M DA-M B-231 cells (350×10³ cell/ml) were seeded in 8-chamber slides (Eppendorf; cat no. 30742036) and incubated for 24 h at 37^°C with 5% CO₂. Subsequently, cells were treated with curcumin (25 µM), B(Cur)₂ (30 µM) and Fe(Cur)₃ (8 µM) or vehicle (control group) for 5 min and immediately fixed with 3.7% formaldehyde at room temperature for 10 min. Cells were counterstained with DAPI at room temperature for 5 min to visualize the nucleus. Stained cells were observed using a BX43 fluorescence microscope (Olympus Corporation; magnification, ×100) to determine the cellular localization of curcumin and its metal derivatives. Images were analysed using CellSens Standard software (version 1.9; Olympus Corporation).

Cell apoptosis was detected by TUNEL assay according to the manufacturer's protocol. ECA109 cells were seeded on coverslips and incubated with serum-free medium for 48 h. Cells were treated with different concentrations of LCL161 (5, 10, and 20 mmol/L) or DMSO for 24 h. After dewaxing and rehydrating with xylene and ethanol, ECA109 cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 h at 25 °C, blocked with 3% H₂O₂ for 10 min, and permeabilized with 0.1% Triton X-100 sodium citrate solution for 3 min. Apoptotic cells were labelled by TUNEL assay, and cell nuclei were labelled with DAPI. Images (magnification × 40) were obtained using a BX43 fluorescence microscope (Olympus Corporation, Tokyo, Japan), and apoptotic cells were analyzed with ImageJ software, each with five randomly selected fields.

Before histology analysis, tissues were immersed in neutral formaldehyde for at least 24 h. Frozen liver tissues were stained with oil red and the pathological scores were graded based on the number and size of stained fat droplets. Other organs which included kidney, intestine, pulmonary, heart and spleen were dehydrated and embedded in paraffin for Hematoxylin-eosin (HE) staining. Afterwards, oil red o staining was conducted with a microtome to acquire a 5 μm serial sections. Sections for immunofluorescence staining were then incubated with ZO-1 or occludin-3 antibody at 4 °C overnights. Then, the sections were washed with PBS for three times and treated with secondary antibody (dissolved in 1% BSA) for 1 h at 25 °C. Images were recorded with an Olympus BX43 fluorescence microscope. To analyse the content of LPS in tissue, intestines were taken and measured with an LPS ELISA kit (Cusabio, CSB-E09945h) following the manufacturer's instructions. Briefly, tissues were pre-treated with 1 × PBS. Reagents were added to samples and incubated for 4.5 h at 37 °C, and the absorbance was read at 540 nm. Standard curve was conducted using known amounts of LPS.