Anti synaptopodin

We have previously described the method to study podocyturia in detail [24 ]. Briefly, a mid-stream freshly voided urine sample was collected on-site after a minimum of 3 h without voiding; and 20 ml of urine were centrifuged at 700g for 5 min in a cytospin. The supernatant was discarded and the sediment was stored in 100 µl aliquots at room temperature mixed with a 1.5 ml solution of 40 % formaldehyde diluted in phosphate-buffered saline (PBS) (pH 7.2–7.4) to reach a final 10 % concentration. Podocyte nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Podocytes were identified by immunofluorescence using anti-synaptopodin as the primary antibody (1:100, Abcam, Cambridge, MA, USA) and IgG anti-rabbit Alexa Fluor^® 488 (1:100, Abcam, Cambridge, MA, USA) as the secondary antibody. Samples were analyzed employing an epifluorescent microscope. Following our standardized technique, podocytes were counted in 10 randomly chosen 20× fields and the average of the counted podocytes in the microscopy fields was considered as the final count for each subject. Results were corrected based on the urinary creatinine concentration of the initial urinary volume of 20 ml employed for podocyte counting [24 , 25 (link)].