Agar low viscosity resin

Cells processed for electron microscopy were treated with wortmannin (50 μM for 2 h or 25 μM for 30 min), BFA (200 μM for 30 min) or DMSO (1 % for 2 h, controls). Cells recovering for 2 h from a 30 min treatment with 25 μM wortmannin were also investigated.
Since mature internodal cells of C. australis are too large for high pressure freezing, chemical fixation was applied as described in Foissner (1991 (link)). Briefly, branchlet internodal cells were fixed for 20 min at room temperature in 1 % glutaraldehyde dissolved in phosphate buffer, pH 6.8. After several washes in buffer, cells were postfixed overnight at 4° C in 2 % OsO₄ dissolved in buffer. Then the cells were dehydrated in an ethanol series at 4° C, and embedded in Agar low viscosity resin (Agar Scientific, Essex, Great Britain) via propylene oxide at room temperature.
Ultrathin sections were stained with uranyl acetate and lead citrate, and micrographs were taken at elastic bright-field mode with a LEO 912 transmission electron microscope equipped with in-column energy filter (Zeiss, Oberkochen, Germany).

Documentation of colony pieces was conducted with a Nikon SMZ25 (Nikon, Tokyo, Japan) equipped with a Nikon DsRi-2 microscope camera, or a Hirox RH2000 microscope. For histological analysis, short branches of the colony were cut off, dehydrated in a graded ethanol series and afterwards infiltrated and embedded into Agar Low Viscosity Resin (Agar Scientific, Stansted, UK). Serial sections were conducted according to Ruthensteiner (2008 (link)). Sections were stained with toluidine blue, documented and analysed with a Nikon NiU microscope.

RPE1 cells were grown at confluence before induction of ciliogenesis for 72 hr by serum deprivation. Cells were fixed 30 min in 2.5% glutaraldehyde (Electron Microscopy Sciences), 2% paraformaldehyde (Electron Microscopy Sciences), 1 mM CaCl₂ in PBS, then washed 3 × 5 min in PBS. Samples were then post-fixed during 30 min in 1% osmium tetroxide (Electron Microscopy Sciences), then washed 3 × 5 min in water. Dehydration was performed using graded series of ethanol in water for 5 min 30, 50, 70, 90, 100, and 100%. Resin infiltration was performed by incubating 30 min in an Agar low-viscosity resin (Agar Scientific Ltd) and EtOH (1:2) mix, then 30 min in a resin and EtOH (2:1) mix followed by overnight incubation in pure resin. The resin was then changed and the samples further incubated during 1.5 hr prior to inclusion in gelatin capsules and overnight polymerization at 60°C. 70 nm sections were obtained using an EM UC6 ultramicrotome (Leica), post-stained in 4% aqueous uranyl acetate and lead citrate, and observed at 80 kV with a Tecnai12 transmission electron microscope (Thermo Fisher Scientific) equipped with a 1K × 1K Keen View camera (OSIS).

Migrating cells were fixed in a 2% formaldehyde/1% glutaraldehyde in PBS solution for 1 hr at room temperature, then washed in PBS. Cells were embedded in a gel of 10% BSA and 10% gelatin. Post fixation was performed in reduced osmium (1% OsO₄ +1% K₃Fe(CN)₆) in water at 4 °C for 1 hr. After extensive washes, cells were incubated in 1% Thiocarbohydrazine in water for 20 min at room temperature and then washed and incubated with 2% OsO₄ in water for 30 min at room temperature. After washes, cells were contrasted with 1% uranyl acetate overnight at 4 °C. The following day, the uranyl acetate solution was removed and the samples washed using pure water. Cells were incubated with a pH 5.5 lead nitrate in aspartic acid solution for 30 min at room temperature, washed in water and dehydrated in successive ethanol baths. Cells were embedded in Agar Low Viscosity Resin (Agar Scientific). 70 nm-thick thin sections were cut using a UC6 ultramicrotome from Leica and deposited on EM grids. Electron microscopy acquisitions were performed using a 120kV Tecnai 12 electron microscope (ThermoFisher) equiped with a OneView 4 K camera (Gatan).

Chemical fixation of branchlet internodal cells of C. australis was as described in Foissner (1991 (link)). Briefly, cells were fixed for 30 min at room temperature in 1% glutaraldehyde dissolved in phosphate buffer, pH 6.8. Following several washes in buffer, cells were postfixed overnight at 4°C in 2% OsO₄ dissolved in buffer. After dehydration in an ethanol series at 4°C, cells were embedded in Agar low viscosity resin (Agar Scientific, Essex, Great Britain). After staining with uranyl acetate and lead citrate, micrographs of ultrathin section were taken at elastic bright-field mode with a LEO 912 transmission electron microscope equipped with in-column energy filter (Zeiss, Oberkochen, Germany).