Immunofluorescent Staining of BKV-Infected Cells

Immunofluorescent staining was performed as previously described [Alcendor 2017] in chamber slide cultures containing mock and BKV-infected GVU cells (podocytes, mesangial cells, and glomerular endothelial cells). Briefly, cells were washed twice with PBS, pH 7.4, air- dried, and fixed in absolute methanol for 20 min at −20 °C. Next, cells were air-dried for 10 min, hydrated in Tris-buffered saline (TBS) (pH 7.6) for 10 minutes, and incubated separately for 1 h with monoclonal antibodies to the BKV major capsid protein VP1 (Santa Cruz Biotech, Temecula, CA, USA), von Willebrand factor (Santa Cruz Biotech), nephrin (Santa Cruz Biotech), and SV40 Large T-Antigen (Abcam), all at a dilution 1:50 in PBS pH 7.4 [30 (link)].