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.

Immunohistochemistry. Tissues were fixed in 4% formaldehyde in PBS and then embedded in paraffin, sectioned, and mounted on slides. Sections were deparaffinized in xylene, rehydrated in graded alcohol, incubated in 1 mM EDTA at 99°C for 20 min, and then rinsed in water. Sections were washed 2 times in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.025% Tween 20), then permeabilized in 0.1% Triton X-100 in PBS for 10 min and washed 3 times in TBST, and then blocked in Trisbuffered saline (TBS) plus 1% BSA plus 5% donkey serum (Sigma). Sections were then incubated for 30 min in anti-NP antiserum diluted in TBS plus 1% BSA, washed in TBST, and then incubated for 30 min in Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody diluted in TBS plus 1% BSA and washed again. The penultimate wash contained 2 g/ml Hoechst 33342. The sections were mounted in Vectashield (Vector Labs), coverslipped, and imaged as described above. Sections were subsequently stained with hematoxylin and eosin and reimaged.