Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease

Stenglein, Mark D.; Sanders, Chris; Kistler, Amy L.; Ruby, J. Graham; Franco, Jessica Y.; Reavill, Drury R.; Dunker, Freeland; DeRisi, Joseph L.

Immunofluorescence. Infected or uninfected cells were plated in collagen-coated glass-bottom culture dishes (MatTek) in cMEM. After 1 day of culture, cells were rinsed twice in phosphate-buffered saline (PBS), then fixed in 4% formaldehyde in PBS for 30 min, and then washed 3 times for 3 min in PBS. Cell membranes were permeabilized in 0.1% Triton X-100 in PBS for 5 min, washed, and then blocked in 1% bovine serum albumin (BSA) for 1 h. Cells were incubated with anti-NP antiserum diluted in blocking buffer for 1 h and then washed 6 times for 4 min in PBS. Cells were then incubated in Alexa Fluor 488-conjugated donkey anti-rabbit antibody (Life Technologies), diluted in blocking buffer for 30 min, and then washed again. The penultimate wash contained 0.5 g/ml Hoechst 33342 (Life Technologies) to stain DNA. Cells were mounted in Vectashield medium (Vector Labs) and imaged. Images were analyzed using ImageJ version 1.43 software (National Institutes of Health). In cases where brightness and contrast were adjusted, this was done so linearly and equally to all images in a set.