Caspase 3

Similar experimental steps with the immunofluorescence, the primary antibody caspase-3 (#PA5-77887, 1:800, ThermoFisher Science, China) was added to culture for overnight at 4 °C. Then the sections were cultured with the Goat anti-Rabbit lgG (H + L) second antibody (#31,460, 1:1000, ThermoFisher Science, China) for 60 min at 37 °C. The sections were stained with the diaminobezidin (DAB) solution at the room temperature. After washing with water, the sections were counterstained with hematoxylin for 60 s. After the dehydration, purification and seal, the results were observed under a light microscope.

The lung tissue from the piglets was extracted using a buffer similar to that used in our previous research. Extracted lung proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bionovas Inc., Toronto, Ontario, Canada) and blotted onto a nitrocellulose membrane. The membrane was treated with SOD2, 3-NT, TNF-α, NF-κB, caspase-3, and β-actin antibodies (Thermo Fisher Scientific Inc.). Secondary antibodies and chemiluminescent substrates (GE Healthcare Life Sciences, USA, Barrington, IL, USA) were also used to induce chemiluminescence that was detected by an LAS-4000 (GE Inc. Healthcare Life Sciences). The optical density of the Western blot was evaluated in ImageJ (version 1.48 t, NIH, Washington, DC, USA).

Paraffin-embedded myocardial tissue sections were dewaxed in xylene and hydrated in ethanol. Tissue sections were then incubated in 30% H2O2 for 30 min. After 10 min of antigen recovery in 10 mm citrate buffer, tissue sections were incubated with anti-TRADD primary antibody (1:100; Thermo Fisher), Caspase 3 (1:1000; Thermo Fisher) and TUNEL (1:500; Thermo Fisher) for overnight. The corresponding mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:500; Thermo Fisher) was then added. The image was observed under an optical microscope.
Serial sections (5 μm) of the heart from each group were selected for immunofluorescence staining. Sections were blocked with 5% goat serum before incubating with rabbit anti-nkx2.5 (nkx2.5, Abeam, 0.5 μg/ml) primary antibodies, followed with incubation with a recombinant terminal deoxynucleotidyl transferase (rTdT) solution for 1 h. Goat anti-rabbit Alexa fluor595 or goat anti-rabbit Alexa fluor532 were used as secondary antibodies for probing TUNEL and goat anti-rat Alexa fluor633 secondary antibody for nkx2.5.

Total RNA was isolated with TRIzol assay (Invitrogen, Pleasanton, CA) following the manufacturer’s protocols. Isolated RNA was reversed transcribed using ABI TaqMan RT kit (Applied Biosystems, Waltham, MA). The pre-designed primer to detect rat miR-153 (Assay ID: 001191), rat Nrf2 (Assay ID: Rn00477784_m1), Bcl-2 (Assay ID: Rn00597992_m1), Bax (Rn01480161_g1), and caspase-3 (Rn00563902_m1) was purchased from Thermo Fisher Scientific. U6 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression were used to normalize gene expression for miRNA and genes mRNA, respectively. Quantitative real-time PCR (qRT-PCR) was performed using an SYBR green/fluorescein or Taqman qPCR Master Mix on an ABI Prism 7500 system (Applied Biosystems).

Fixed brain samples were incubated with polyclonal antibodies against t-tau, whereas incubation with phosphate-buffered saline (PBS) served as a negative control. DAPI was used for nuclear staining. Microphotographs were acquired using a Motic Pathology slide scanner (Meyer Instrument, Houston, TX, USA). Also, brain sections were stained with NeuN (GeneTex, San Antonio, TX, USA), the cell apoptosis marker of activated caspase-3 (Cell Signaling Technology, Beverly, MA, USA), and nuclear-staining DAPI (Sigma-Aldrich, St. Louis, MO, USA). For neuron detection, a Nissl staining kit (MDS Analytical Technologies, Sunnyvale, CA, USA) was used to measure Nissl bodies in the cytoplasm of neurons. A fluorescent microscope (EVOS FL imaging system; Thermo Fisher Scientific) was used to obtain NeuN, caspase-3, and DAPI images. Automated microscopy (Tissuefaxs; TissueGnostics, Vienna, Austria) was used to obtain Nissl-stained images. The regions of interest were selected (n = 3 sections per region) based on significant changes and our previous reports [7 , 70 ]. All images were taken under the same exposure time.

For cell proliferation assay, the mice with indicated genotype were given 2 mg of BrdU i.p. 3 hr before sacrifice. Cells were first stained with surface markers and then were fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (554714, BD Biosciences). Next, cells were intracellularly stained with anti-BrdU antibody using the BrdU Flow Kit (BD Biosciences) according to manufacturer’s introduction. For apoptosis assays, the cells were first stained with surface markers, followed by staining with Caspase-3 (Thermo Fisher Scientific) at 37℃ for 1 hr.

A549 and U-251 MG cells were seeded at a density of 1 × 10⁶ cells per 15-cm² plate the day before infection. Cells were infected with ZIKV at indicated MOI as described above and harvested at the indicated time points. Prior to fixation, cells were stained with Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cells were fixed using Cytofix/Cytoperm (BD Bioscience, Mississauga, ON, Canada) for 20 min and then stained with anti-J2 dsRNA (Scicons, Szirák, Hungary) diluted 1:1000, anti-4G2 (Millipore, Etobicoke, ON, Canada) diluted 1:200, or anti-cleaved Caspase-3 (Cell Signaling, Danvers, MA, USA) diluted 1:800 in PermWash (BD Bioscience) for 1 h followed by secondary staining with goat anti-mouse Alexafluor 488 or goat anti-rabbit Alexafluor A546 (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:300 for dsRNA or 1:500 for 4G2 and Caspase-3 in PermWash. Data were acquired on an LSRFortessa Analyzer (BD Bioscience) with BD FACSDiva software. Data analysis was performed using FlowJo software version 10.5 (BD Bioscience, Mississauga, ON, Canada). Debris and doublets were excluded from the analysis using forward-scatter width discrimination, and the percentage of dsRNA-, 4G2-, or Caspase-3-positive cells was determined by comparison to mock-infected cells.