Evos fl

To monitor ZIKV infection in cells, supernatant was removed at the indicated times and cells were rinsed in PBS. Cells were fixed in PBS–4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 15 min at room temperature (RT), and permeabilized with PBS–0.1% Triton (Sigma) for 3 min at RT. Non-specific sites were blocked with PBS–1% bovine serum albumin (BSA, Sigma)–0.1% Tween 20 (Sigma) for 30 min at RT. ZIKV envelope protein (E) staining was performed using a primary mouse anti-flaviviral E antibody (4G2; home-purified from the ATCC hybridoma [30 (link)]) diluted in PBS–0.2% BSA–0.2% Tween 20 for 1 h at RT, and a secondary Alexa Fluor 488-coupled goat anti-mouse antibody (Life technologies) diluted in PBS–0.2% BSA for 30 min at RT. Finally, cells were mounted in Fluoromount G–DAPI (SouthernBiotech, Birmingham, AL, USA) and imaged on a fluorescence microscope (EVOS FL, Life Technologies). Cells were washed twice with PBS between each step.

Viable cells were detected at day 1 post-seeding by intracellular esterase activity using Calcein-AM. Briefly, cells were incubated with 2 µM Calcein-AM (Biomol, Hamburg, Germany) and Hoechst 33342 (Sigma, Munich, Germany) at room temperature (protected from light) for 30 min. Following a washing step with PBS, images were taken using an EvosFL epifluorescence microscope (Life Technologies, Darmstadt, Germany).

Frozen tissue sections and paraffin tissue sections were used for immunofluorescence staining. Paraffin sections were traditionally fixed, regularly dewaxed, repaired under high pressure, and closed. After sealing, samples were stained with the primary antibody: F4/80 (1:1,000, Cell Signaling Technology) at 4°C overnight, and then incubated with the fluorescent secondary antibody: anti-rabbit IgG-fluorescein isothiocyanate (IgG-FITC) (1:1,000, Cell Signaling Technology) according to the manufacturer’s instructions. All images were observed using a fluorescence microscope (EVOS FL, Life Technologies, Carlsbad, CA).

The mitochondrial membrane potential was analyzed using flurochrome reporter JC-1 (1μM) dye (5,5,6,6-tetra-chloro-1,1,3,3-tetra-ethyl-benz- imidazolo-carbocyanine iodide) (BD Bioscience) as per manufacture's instruction. In brief, cells were treated with 3,4-diarylpyrazoles (50 μM) for 24 h and then cells were incubated with JC-1 dye (1μM) for 10 min. Photographs were taken using an inverted fluorescence microscope (EVOS FL, Life technologies) and imaged first under red filter and then under green filter. Red fluorescence indicated cells with intact mitochondria while green fluorescence indicated cells with depolarized mitochondria. Values are expressed as total red CTCF from three replicates.

U87 tumor spheroids were established as previously described (Li et al., 2014 (link)). For spheroid formation, U87 cells (2 × 10⁵ cells/ml) were seeded in an ultralow attachment plate (Corning, USA) in DMEM medium. Then, the tumor spheroids (500 μl) were picked into a 6-well plate coated with 2% agarose (m/v) and incubated with Fe(PIP)₃SO₄ for 12 h. To determine the penetration ability of Fe(PIP)₃SO₄, tumor spheroids were first rinsed with PBS and then scanned by consecutive layers from top to middle zone via confocal laser scanning fluorescent microscope (Carl Zeiss, Germany). The bulk of U87 tumor spheroids was calculated using a fluorescence microscope (EVOS^® FL, Life Technologies, USA) in white light at different time points (0, 1, 2, 3, and 5 days), the formula V = (π × d_max × d_min)/6, R = (V_i/V₀) ×100%, as previously reported (Mo et al., 2016 (link)).

After treatment, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and fixed with 4% (w/v) paraformaldehyde for 10 min at room temperature. This was followed by permeabilization with 0.2% Triton-X-100 solution for 20 min at room temperature and treatment with 2% (w/v) paraformaldehyde for 10 min at room temperature. Unspecific binding sites were blocked with 5% (w/v) BSA for 1 h at room temperature, followed by incubation with the first antibody overnight at 4 °C (Table 1). After washing three times with PBS, cells were incubated with Alexa-fluor labeled secondary antibody (1:1000) for 2 h at room temperature (Table 1). Nuclei were stained with Hoechst 33,342 (1:1000). Images were taken with an epifluorescence microscope (EVOS FL, life technologies, Darmstadt, Germany). Pictures were analyzed with the ImageJ software (Version 1.5, NIH, Bethesda, MD, USA) by two independent investigators in a blinded fashion. Based on the microscopic pictures taken, cilia length was determined by the maximum intensity projection method [58 (link)].