The measurement of autophagy using transmission electron microscopy

HK2 cells grown on glass coverslips were transfected with KCa3.1 or scrambled siRNA overnight and then incubated with TGF-β1 for 48 hours as described above. The samples were prepared for transmission electron microscopic analysis as previously reported⁴⁸. Briefly, cells were washed with pre-warmed PBS twice, and then fixed in 2% glutaraldehyde in PBS for 60 minutes at room temperature. Fixed cells were washed with PBS 3 times then postfixed with 1% in osmium tetroxide in PBS for 1hr. After rinsing 3 times with distilled water, the samples were further stained with 1% tannic acid for 1 hour. Finally, the cells were infiltrated and double-embedded in Epon. Sections of 70 nm were generated with an ultramicrotome (Ultracut 7, Leica) and post-stained with 2% aqueous uranyl acetate and Reynold’s lead citrate for 10 min each. The specimens were examined with the JEOL 2100TEM at 200 kV.