Antifade mounting medium

Dimethyl sulfoxide (DMSO) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Proteinase K was bought from Tiangen Biotech Co., Ltd. (Beijing, China). Antifade Mounting Medium was bought from Boster Biological Technology Co., Ltd. Paraformaldehyde fix solution at a concentration of 4% was obtained from Servicebio Technology Co., Ltd. (Wuhan, China). An RAA fluorescence kit was obtained from Jiangsu Qitian Gene Biotechnology Co., Ltd. (Wuxi, China). A Wizard^® Magnetic DNA Purification System for Food was purchased from Promega Biotech Co., Ltd. (Madison, WI, USA). All primers and probes were synthesized by Sangon Biotech (Shanghai, China). Skim milk was purchased at the local supermarket (Rainbow, China). TOMA was provided by Ningbo International Travel Healthcare Center (Ningbo Customs Port Outpatient Department). The final concentration of TOMA dissolved in 20% DMSO was 1 mg/mL. It was stored at −20 °C in the dark for further use.

Cells were cultured on coverslips in 24 well plates and were sequentially treated with 4% paraformaldehyde solution (Solarbio, Beijing, China) for 40 min, 0.1% Triton-X (Solarbio, Beijing, China) for 20 min, and goat serum (Boster, Wuhan, China) for 1 h. Afterwards, cells were incubated with a mixture of LC3B and LAMP1 primary antibodies overnight at 4°C, incubated with a secondary antibody mixture for 2 h at room temperature, and stained with DAPI for 5 min. Finally, slides were mounted with antifade mounting medium (Boster, Wuhan, China) and observed under a confocal laser scanning microscope (Leica, German). Cells without primary antibodies incubation were used as a negative control.

Placental tissues were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Serial sections (3 μm) of paraffin-embedded tissues were analyzed by IF as described elsewhere.²³ (link) Briefly, tissue slides were deparaffinized in xylene, rehydrated in a serial ethanol gradient, and blocked with 3% H₂O₂ for 10 min. The slides were then immersed in TE buffer (10 mM Tris and 1.0 mM EDTA, pH 9.0), warmed in a microwave oven at 92°C–98°C for 15 min for antigen retrieval, and cooled to room temperature. The slides were then blocked with 10% goat serum (Boster, China) for 1 h at room temperature, incubated with primary antibodies overnight at 4°C, and then incubated with fluorescence-labeled secondary antibodies (Bioservice, China) at 37°C for 1 h. The nuclei were subsequently counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Boster, China) and mounted with antifade mounting medium (Boster, China). The cells were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and blocked with 10% goat serum (Boster, China). After overnight incubation with primary antibodies at 4°C, the cells were incubated with fluorescence-labeled secondary antibodies (Bioservice, China) at 37°C for 1 h. The nuclei were stained with DAPI, and images were captured with an EVOS microscope (Life Technologies, USA) and/or confocal microscope (Zeiss, Germany).

HCT-8 cells transfected with EGFP-LC3 were cultured in 24-well plates containing glass slides (2 × 10⁵ cells/well). At 24 h post-transfection, the miR-26a-mimic or miR-30a-inhibitor was transfected into the cells; cells were cultured with ERK inhibitor (LY3214996, 1 μM; Selleckchem, USA), P38 inhibitor (SB203580, 5 μM; Selleckchem, USA) or Rapamycin (1 μM, Selleckchem, USA) for 1 h. The cells were incubated with sporozoites (sporozoite:cell = 1:2) for 12 h before harvesting. Cells on glass slides were washed with PBS three times (5 min for each time), fixed with 4% paraformaldehyde for 15 min at room temperature (RT) and treated with 0.25% TritonX-100 (Life Technologies Corporation, USA) for 20 min at RT. Then, the cells were blocked with 3% Bovine Serum Albumin (Boster Biological Technology, USA) in PBS for 30 min at RT and incubated with a 1:100 dilution of the C. parvum virus capsid antibody (prepared in our laboratory) overnight at 4 °C. The cells were washed and incubated with CoraLite594 secondary antibody (Proteintech, Wuhan, China) for 1 h at RT. At last, the cells were sealed by Antifade Mounting Medium (BOSTER Biological Technology, USA) with 0.1% DAPI (Thermo Science, Waltham, MA) and observed under a laser scanning microscope (Zeiss LSM710, Germany).

HepG2 cells (4 × 10⁴/well) were cultured on coverslips in 24-well plates for 24 h and subsequently treated with 20 μM of fisetin for 6 h. After treatment, cells were fixed using 4% paraformaldehyde solution (Dingguo, Beijing, China) for 40 min, permeabilized with 0.1% Triton-X (Solarbio, Beijing, China) for 20 min and blocked with goat serum (Boster, Wuhan, China) for 60 min. Then, cells were incubated overnight with Nrf2 primary antibody at 4 °C. Then, cells were incubated with fluorescent secondary antibody for 2 h at room temperature and stained with DAPI for 5 min. Finally, coverslips were mounted using antifade mounting medium (Boster, Wuhan, China) on slides, observed, and photographed under a fluorescence microscope (ZEISS, Jena, Germany).

For immunofluorescence staining, cells were plated on coverslips and fixed with a 4% paraformaldehyde solution for 15 min at room temperature. After fixation, cells were washed with PBS and permeabilized with PBS containing 0.5% Triton X-100 for 30 min. Cells were incubated with 4 M HCl for 15 min to denature the DNA, rinsed with distilled water, and placed in 100 mM Tris-HCl (pH 8.5) for 10 min. After washes with PBS, non-specific binding sites were blocked with blocking buffer (10% FCS and 0.1% Tween-20 in PBS) for 1 h. Next, cells were incubated with 5mC (1:100, Cell Signaling Technology, Danvers, U.S.A.) or 5hmC (1:100, Cell Signaling Technology, Danvers, U.S.A.) overnight at 4°C. Subsequently, cells were washed with PBS three times for 10 min each, followed by an incubation with Alexa Fluor 488- or 594-conjugated secondary (anti-mouse or anti-rabbit) antibodies for 1 h at room temperature. Cells were washed three times with PBS for 10 min each, and the coverslip was mounted on a glass slide using an anti-fade mounting medium (BOSTER, China). Cells were imaged using a fluorescence microscope. Image analysis software (ImageJ) was used to evaluate the average fluorescence intensity (AFI) in the cells.

HepG2 cells (4 × 10⁴/well) were cultured on coverslips in 24-well plates for 24 h. Then cells were treated with prodigiosin (0.4 μM) for 6 h and MC-LR (1 μM) for 1 h. After treatment, cells were fixed using 4% paraformaldehyde solution (Dingguo, Beijing, China) for 40 min, permeabilized with 0.1% Triton-X (Solarbio, Beijing, China) for 20 min, and blocked with Goat Serum (Boster, Wuhan, China) for 1 h. Then, cells were incubated with Nrf2 primary antibody (Abcam, Cambridge, UK) at 4 °C overnight. After that, cells were incubated with fluorescent secondary antibody for 2 h at room temperature and stained with DAPI for 5 min. Finally, coverslips were mounted using antifade mounting medium (Boster, Wuhan, China) on slides, observed, and photographed using fluorescence microscope (ZEISS, Jena, Germany).