Mounting medium for fluorescence

CPE positive and uninfected (mock) Hep-2 cells were digested by 0.02 % EDTA, spotted to eight well glass slide (MP Biochemicals, CA, USA) separately. The slides were fixed with cold acetone after air drying the cells. After blocking with 3 % BSA for one hour at room temperature, the slides were incubated with sheep anti-HRSV IgG antibody (1:500, Millipore) at 37 °C for one hour followed by washing three times with PBS, the slides were reacted with Alexa Fluor 488 labelled anti-sheep IgG (1:500, Invitrogen) at room temperature for one hour. After additional three washes with PBS, the slides were air dried, sealed with mounting medium for fluorescence (Vector Laboratories, Inc.). The samples were subsequently examined using a fluorescence microscope.

For RPE explant flat mount, tissues were stained with PI solution at 100 μg/ml, then were fixed with 4% PFA followed by phalloidin and DAPI staining. Tissues were then mounted in mounting medium for fluorescence (Vector laboratories, Burlingame). Pictures were taken using a fluorescence microscopy. Tissues were also collected and embedded in OCT (Fisher Healthcare, 4585) for cryosection and immunofluorescence staining. Tissues were incubated with cold acetone and permeabilized with 0.25% Triton-X/0.05% NaN3 in PBS. After blocking with blocking buffer (1x PBS containing 0.5% Triton-X100 and 5% horse serum), the tissues were then incubated with primary antibodies overnight at 4 °C. After wash with 1x PBST solution, tissues were incubated in secondary antibody conjugated with fluorescence for 2 h (1:500). Nuclei were outlined by DAPI. Tissues were then mounted in mounting medium for fluorescence (Vector laboratories, Burlingame). Primary antibodies: anti-pRIPK3 (mouse): Cell Signaling, 91702S, anti-pMLKL (mouse): Cell Signaling 37333S, anti-RPE65: Abcam, ab231782, Phalloidin: Cell Signaling, 8878S. Secondary antibodies: Alexa fluor 594 goat anti-rabbit IgG (Invitrogen, A11012), Alexa Fluor 488 goat-anti-rabbit IgG (Life technologies, A11008).

Cells were seeded on 0.1% gelatin coated coverslips for immunofluorescence staining. Control and treated cells were fixed in 4% paraformaldehyde (PFA) for 30 min, then were washed with 1x PBS and permeabilized in 1x PBS containing 0.5% Triton-X100. After 1 h incubation in 1x PBST solution (1x PBS containing 0.1% Tween-20) containing 5% horse serum, cells were exposed to 1x PBST diluted primary antibody and incubated overnight at 4 °C. Cells were then washed with 1x PBST solution and incubated in secondary antibody conjugated with fluorescence for 2 h (1:500). Nuclei were outlined by DAPI. Coverslips were mounted in mounting medium for fluorescence (Vector laboratories, Burlingame). Primary antibodies: anti-pRIPK3: Cell Signaling, 93654S, anti-pMLKL: Abcam, ab187091, anti-ZO-1: Invitrogen, MA3-39100-A647. Secondary antibodies: Alexa fluor 594 goat anti-rabbit IgG (Invitrogen, A11012), Alexa Fluor 488 goat-anti-rabbit IgG (Life technologies, A11008). Fluorescent intensity was measured using ImageJ, data was normalized by dividing the intensity of the signals of pRIPK3 or pMLKL by the fluorescence intensity of DAPI.