1) was applied. The slide was allowed to sit at room temperature until the droplet applied was completely spread across the entire cover slip area, and then the cover slip was sealed using Valap (1:1:1 vaseline, lanolin, paraffin wax) to avoid evaporation. Samples were covered with Vactosertib in vivo aluminum foil to reduce photobleaching by stray light until imaging. Preparation of Oleic Acid Vesicle Samples ~10 mM oleic acid vesicles containing Smoothened Agonist ic50 5′-6-FAM-labeled RNA (5′-CCAGUCAGUCUACGC-3′) were prepared by mixing 1.6 μL pure oleic acid (3.17 M) with 50 μL of 10 μM RNA in 500 μL 180 mM bicine buffer adjusted to pH 8.5 with NaOH, followed by vortexing
for 30 s. The sample was covered with foil and allowed to gently tumble overnight. A 3 μL droplet was applied to a glass slide as above for microscopy. The
glass slide was then allowed to sit (cover slip down) at room temperature for 30 min to allow larger vesicles to rest on the surface of the cover slip. Preparation of a Dextran/PEG ATPS Inside Oleic Acid Vesicles To 840 μL of 5.95 % PEG 8 kDa, 10.7 % Dextran 10 kDa, 200 mM bicine pH 8.5 (adjusted with NaOH), 0.5 μL 200 mM HPTS (8-hydroxypyrene-1,3,6-trisulfonate, stock in H2O, 0.12 mM final concentration) and 10 μL of 100 μM RAD001 order 5′-Cy5-labeled RNA (5′-GCGUAGACUGACUGG-3′ in H2O, 1.2 μM final concentration) were added. The solution was vigorously vortexed and visually inspected to verify that it contained only one phase. Subsequently, 3 μL of oleic acid were added to the solution and after another vigorous vortexing, the solution was tumbled over night on a rotating wheel (6 rpm) to allow vesicle formation. The Histidine ammonia-lyase next day, the vesicles were purified from unencapsulated dye and RNA using a short 1 cm Sepharose 4B gel filtration column and 1 mM
oleic acid in 200 mM bicine (adjusted to pH 8.5 with NaOH) as a running buffer. 6 μL of gel-filtered vesicles were spread out (to around 1 cm2) on a 25×75 mm microscope slide and the droplet was allowed to evaporate for 6 min at room temperature. Then an 18x18mm coverslip was placed onto the droplet and the slide was sealed using Valap. Alternatively, a 3 μL droplet was placed on a slide and a coverslip was placed immediately on top of it. In this case, the coverslip was not sealed, but only fixed in the corners with Valap, and evaporation was allowed to occur through the edges over several hours. Slides were observed either with a confocal microscope (see below) or with a Nikon (Tokyo, Japan) TE2000 inverted fluorescence microscope with a 100× oil objective. Fluorescence Recovery After Photobleaching (FRAP) by Confocal Microscopy Each sample was imaged using a confocal microscope at 488 nm (pinhole 1 AU). Confocal microscopy was performed using a Leica (Solms, Germany) SP5 AOBS Scanning Laser Confocal Microscope (63×, 1.4-0.6 N. A.