Uranyl acetate replacement stain

One sponge individual from 1 day, with its corresponding three differently-treated specimens, was analysed by TEM in order to characterise the spherulous cells observed by light microscopy. Embedded samples were cut into ultra-thin (70 nm) sections using an ultramicrotome (Reichert Ultracut S, Leica, Austria). Sections were mounted on pioloform coated grids and contrasted with uranyl acetate replacement stain (Science Services, Germany) for 20 min and subsequently with Reynold´s lead citrate for 3 min. Ultra-thin sections were imaged with a Tecnai G2 Spirit BioTwin transmission electron microscope (80 kV, FEI, USA) at the Central Microscopy of University of Kiel (Germany).

Carbon-coated copper grids, 200 mesh (Science Services, Munich, Germany) were treated with oxygen plasma (Zepto, Diener electronic GmbH, Ebhausen, Germany). After this, 3 µL of precipitation-purified rAAV sample [25 (link)] was applied to the grid and incubated for 2 min. Excess liquid was drained off, the grid was dried at room temperature and washed with three drops of distilled water. Negative staining was performed using 3 µL 2% (v/v) uranyl acetate replacement stain (Science Services) for 30 s. Excess liquid was drained off and grids were dried before channeling the sample into the microscope. rAAVs were visualized with a CM100 (PW6021) instrument (Philips, Hamburg, Germany) with an acceleration voltage of 80 kV. Images were analyzed using the Soft Imaging Viewer (Olympus, Münster, Germany) and ImageJ [50 (link)].