Slowfade gold antifade mountant with dapi

OVCA cell lines were plated in 8-well chamber slides (Nunc, USA) at 3000 cells/well and were incubated overnight in 5% CO₂ at 37°C. Cells were subsequently treated with SP-6-27 (0.5 μM) or DMSO vehicle control. After 24 h, cells were washed with PBS, fixed with 4% paraformaldehyde for 25 min at room temperature (RT) and permeabilized with methanol: acetone (9:1) for 5 min at −20°C. Slides were blocked with image-iT FX Signal Enhancer (Thermo Fisher Scientific) for 45 min at RT. Primary antibody staining for alpha-tubulin (1:250), beta tubulin (1:250) and caspase-3 (1:250) [Abcam, MA, USA] was performed by incubating the slides for 2 h at RT. The cells were incubated with secondary antibody Alexa Fluor^® 488-conjugated goat donkey-anti Rabbit (Invitrogen, USA) for 45 min at RT. The cells were prepared for viewing using SlowFade® Gold Antifade Mountant with DAPI (Invitrogen). For confocal microscopy, the stained cells were imaged by Fluoview Fv10i confocal microscope (Olympus). Analysis was performed using Fv10i Flouview Ver.3.0 software. Experiments were repeated at least twice in triplicate.

Immunofluorescence was performed as previously described [31 (link)]. Slides were de-paraffined using xylene and then treated with a graded series of ethanol washes. Sections were heated in citric acid buffer (pH 6.0, 100 °C, 10 min) for antigen retrieval and subsequently blocked with 5% bovine serum albumin in PBS containing Tween (PBST) for 1 h (room temperature) prior to an overnight incubation (4 °C) with the primary antibodies diluted in PBST.
Sections were then incubated with secondary antibodies at room temperature (1 h). Sections were stained using SlowFade^® Gold Antifade Mountant with DAPI (Invitrogen, Carlsbad, CA, USA) and a coverslip applied. Antibodies are listed in Table A2.
For cell staining, cells were cultured in a 12-well plate on glass coverslips until ~50% confluence, at which time they were fixed using 4% paraformaldehyde (30 min, room temperature) and processed as described above.
Digital fluorescence images were captured, and positive cells were counted manually using ImageJ software (NIH, version 1.8.0). To determine the number of positive cells, four sections were chosen at random, cells were stained with DAPI, and signals of interest were counted and averaged. For the analysis of macrophage subtypes, the percentage of dual-positive cells to CD68 positive cells was used.

MRC-5 cells were grown on poly-L-lysine-coated coverslip, fixed with 4% paraformaldehyde for 10 min and rinsed with cold PBS. Then, cells were permeabilized in 0.2% Triton X-100-contained PBS for 10 min and blocked with 3% BSA in permeabilizing buffer. Cells were incubated with anti-HCoV-OC43 spike protein antibody (CusaBio), or anti-HCoV-OC43 nucleocapsid protein antibody (Merck) at 4 °C overnight. Cells were washed with cold PBS three times and incubated with AlexaFluor488-conjugated goat anti-rabbit IgG antibody (Cat. No. A11001, Thermo Fisher Scientific Inc.) or AlexaFluor555-conjugated goat anti-mouse IgG antibody (Cat. No. A21428, Thermo Fisher, Waltham, MA, USA) for 1 h at room temperature. These cells were washed with PBS and mounted using SlowFade™ Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA) and visualized using an Olympus IX71 fluorescence microscope (Olympus Corporation, Tokyo, Japan) and the cellSens program (Olympus).

The testes dissected from mice was fixed in 4% paraformaldehyde for 24 h and then incubated in sucrose-PBS solution (10% sucrose for 1 h, 20% sucrose for 1 h, and 30% sucrose overnight) at 4 °C. Frozen sections were cut to a thickness of 7 μm using the Leica CM1950 (Leica, Solms, Germany). Cryosections were washed in PBS and permeabilized with PBS containing 0.5% Triton X-100. The slides were blocked with 5% BSA and then incubated with primary antibody at 4 °C overnight. AlexaFluor™ conjugated secondary antibody was added for 1 h at room temperature. Finally, the slides were mounted with SlowFade™ Gold Antifade Mountant with DAPI (Invitrogen) and observed under LSM 780 confocal microscope (Zeiss, Germany) for fluorescent signal analysis.

The upper right lobe of each lung was inflated with 4% PFA and processed routinely for light microscopy with hematoxylin and eosin stain. Morphometric analysis was performed in a blinded manner using ImageJ software (version 1.50e; NIH). The percentage of total lung area involved with inflammation was calculated by dividing the cumulative area of inflammation by the total lung surface area for each sample.
Immunofluorescence was performed on formalin-fixed lung sections as described previously (65 (link)). Sections were probed using a purified rabbit polyclonal anti-CD3e (1:100; ab185811, Abcam), and visualized by adding Alexa Fluor 568 goat anti-rabbit (1:500; A-11011, Invitrogen). SlowFade Gold Antifade Mountant with DAPI (Invitrogen) was used to counterstain tissues and to detect nuclei. Representative images were obtained using an Olympus BX61 microscope and were recorded using a digital camera (DP70, Olympus) using the Olympus cell^P software.