Fv1000 flowview confocal microscope

HeLa cells seeded onto glass coverslips were infected with MAYV at a multiplicity of infection (MOI) of 1. At 24 h after infection, cells were fixed in 2% paraformaldehyde for 20 min, and permeabilized with 0.25% Triton-X100. Cells were then incubated in 2% bovine serum albumin solution in PBS for 20 min and stained overnight at 4 °C with a mouse immune ascitic fluid anti-MAYV antibody (kindly provided by Dr. Scott Weaver, WRCEVA, UTMB, USA). Finally, cells were washed with PBS buffer and incubated with a Goat anti-mouse secondary antibody, Alexa-Flour 488 conjugate (Invitrogen, Carlsbad, CA, USA), for one hour in the dark. Coverslips were mounted on slides with Prolong Diamond Antifade Mountant with Dapi (Invitrogen, Carlsbad, CA, USA), and the images were obtained with a FV1000 Flowview Confocal microscope (Olympus, Lombard, IL, USA). The pictures were exported and analyzed with ImageJ software [47 (link)]. The number of MAYV-positive cells were counted in at least 10 fields and represented as the percentage of positive cells.

Vero or HeLa cells cultivated on glass coverslips were infected with CHIKV, MAYV or UNAV at an MOI of 1. Following 24 h of infection, cells were fixed with 2% paraformaldehyde in PBS buffer for 20 min and permeabilized with 0.25% Triton-X100. Then, the cells were blocked in 2% bovine serum albumin solution in PBS for 20 min and stained overnight at 4 ºC with anti-CHIKV, anti-MAYV or anti-UNAV mouse ascitic fluid (kindly provided by Dr. Scott Weaver, WRCEVA-UTMB, USA). Afterward, cells were incubated with a Goat anti-mouse secondary antibody (Alexa Flour 488, Invitrogen, Carlsbad, CA, USA) for 1 h in the dark. Finally, coverslips were mounted on slides with Prolong Diamond Antifade Mountant with Dapi (Invitrogen, Carlsbad, CA, USA), and the images were captured with an FV1000 Flowview Confocal microscope (Olympus, Lombard, IL, USA). The pictures were analyzed with ImageJ software. The number of virus-positive cells were estimated in at least 10 fields and denoted as the percentage of positive cells.

Immunofluorescence experiments were performed as previously reported [22 (link)]. Briefly, HDFs grown on glass coverslips were infected with MAYV at an MOI of 1 and treated with the inhibitors or a control. After 24 h of infection, cells were fixed, permeabilized, blocked, and stained overnight at 4 °C with an anti-MAYV mouse ascitic fluid (kindly provided by Dr. Scott Weaver, WRCEVA-UTMB, Galveston, TX, USA). Next, cells were incubated with a goat anti-mouse secondary antibody (Alexa Flour 488 or Alexa Flour 568, Invitrogen, Carlsbad, CA, USA) for 1 h in the dark. Lastly, coverslips were mounted on slides with Prolong Diamond Antifade Mountant with Dapi (Invitrogen, Carlsbad, CA, USA), and images were acquired with an FV1000 Flowview confocal microscope (Olympus, Lombard, IL, USA). The images were analyzed with ImageJ software.