Choanoflagellate transfection illuminates their cell biology and the ancestry of animal septins

David S. Booth; Heather Szmidt-Middleton; Nicole King

Epifluorescence and differential interference contrast images were recorded using a Zeiss 520 Axio Observer.Z1/7 Widefield microscope with a Hamamatsu Orca- The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/343111 doi: bioRxiv preprint Cells were fixed in two stages. First, the coverslip was washed once with 6% acetone in 547 cytoskeleton buffer (10 mM MES, pH 6.1; 138 KCl, 3 mM MgCl2; 2 mM EGTA; 675 mM 548 Sucrose), which better preserves the actin cytoskeleton (Cramer and Mitchison, 1995; Symons 549 and Mitchison, 1991), and then incubated for 10 min at room temperature after a second 550 application of the acetone solution. Subsequently, the coverslip was washed once with 4% 551 formaldehyde diluted in cytoskeleton buffer and then incubated for 15 min at room temperature 552 after a second application of the formaldehyde solution. Last, the coverslip was gently washed 553 three times with cytoskeleton buffer. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/343111 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/343111 doi: bioRxiv preprint 1 2 2 3 4