Model lsm710

Cells cultured on Lab-Tek II chamber slides were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 2% BSA for 30 min. Cells were then incubated with anti-LC3 antibody (Cell Signaling, Beverley, MA, USA) overnight followed by FITC-conjugated anti-IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 1 h. DAPI was used for counter-staining. Images were taken with a laser-scanning confocal microscope (Model LSM710, Zeiss, Jena, Germany). LC3 morphology was assessed by an independent viewer in a blind manner. For each independent experiment, 5–10 random high-power fields (at least 50 cells in total) were surveyed. Cells were arbitrarily categorized into punctate LC3+ and LC3- groups (as guidance, LC3 punctation induced by amino-acid starvation was used as a positive control). We also counted the average number of LC3 puncta in individual cells.

RAW264.7 or differentiated THP-1 cells were cultured on Lab-Tek II chamber slides (Thermo Scientific, Pittsburgh, PA, USA) and loaded with DiI-Ac-LDL (30 µg ml⁻¹) for 2, 4 or 8 hr. Then cells were fixed with 4% paraformaldehyde at room temperature for 20 min. After 3 rinses, cells were counterstained with DAPI. Fluorescent images were obtained with a confocal microscope (Model LSM710, Zeiss, Jena, Germany) at 549 nm excitation and 565 nm emission. To assess macrophage transformation into foam cells, we analyzed the accumulation of intracellular lipid droplets using Oil Red O staining. Briefly, cells cultured on slides were loaded with ox-LDL (80 µg ml⁻¹) for 48 hr, then cells were fixed with 4% paraformaldehyde, and stained with Oil Red O (0.3% dissolved in isopropyl alcohol) for 15 min at room temperature. Hematoxylin was used for counterstaining. Slides were evaluated by light microscopy.

For microscopic visualization of A. hydrophila biofilm formation, the A. hydrophila cells could form biofilm on glass slides (1 × 1 cm) placed in 24 wells MTP supplemented with and without NA (93.75–750 μg/ml), and they were incubated for 24 h at 30°C in static condition. After incubation, the glass slides were washed with distilled water and processed as follows.
For light microscopic visualization, the glass slides were washed with distilled water and stained with a 0.4% CV solution for 3 min. The stained-glass slides were then washed with distilled water, air dried, and then mounted on a microscopic slide with the biofilm directed upwards and imaged using a light microscope (Nikon Eclipse Ti 100, Tokyo, Japan) at a magnification of ×400 (Sivaranjani et al., 2018 (link)).
For confocal laser scanning microscopic (CLSM) analysis, the glass slides were washed with distilled water and stained with 0.1% acridine orange solution (w/v) (Sigma, St. Louis, MO, United States) for 1 min. The stained-glass slides were washed with distilled water and air dried at room temperature. Then, the stained slides were imaged using CLSM (Model LSM 710, Carl Zeiss, Germany) at ×200 magnification (Bakkiyaraj and Karutha Pandian, 2010 (link)).