S150b sputter coater

To study cell morphology on glass coverslips and electro-spun scaffolds, the samples were fixed and then dehydrated. First, culture media was removed, and samples were washed with PBS twice for two minutes. Then, cells were fixed using 2.5% glutaraldehyde (Merck KGaA, Darmstadt, Germany) in PBS for 1 h. The glutaraldehyde solution was removed, and samples were washed 3 times for 15 min with PBS. Next, the samples were washed in distilled water for 5 min before sequential dehydration using ethanol (Sigma Aldrich, Gillingham, UK). Samples were placed in increasing concentrations of ethanol in distilled water for 15 min each from 35% (v/v) to 60%, 80%, 90%, and 100%. Next, the samples were treated with ethanol: hexamethyldisilazane (HMDS) (Sigma Aldrich, Gillingham, UK) 1:1 (v/v) solution for 1 h. Finally, samples were treated twice with 100% HMDS for 5 min before they were left to air-dry for 1 h prior to gold-coating (Edwards S150B sputter-coater) and SEM imaging (Tescan, Vega3).

For scanning electron microscopy, ift88 homozygous mutant and phenotypically wild-type sibling larvae at 4 dpf were fixed overnight in 2.5% glutaraldehyde/0.1M sodium cacodylate buffer. Samples were washed in buffer, post-fixed in 2% aqueous osmium tetroxide for 1 h, washed in buffer again and then dehydrated through a graded ethanol series (50, 75, 95, 100%) before being dried in a mixture of 50% hexamethyldisilazane (HMDS) in 100% ethanol. Final drying was in 100% HMDS. After removal of the final HMDS wash, samples were left to dry in a fume hood overnight. Samples were mounted onto a pin stub using a Leit-C sticky tab and Leit-C mounting putty, gold-coated using an Edwards S150B sputter coater, and examined in a Tescan Vega3 LMU Scanning Electron Microscope at an operating voltage of 15 kV and imaged using a secondary electron detector.

Scanning electron microscopy (SEM) was used to visualise the appearance and coverage of biofilm developed on the PWG coupons inserts (Figure 1). The inner surface (insert) of 3 PWG coupons (total area 90 mm²) was collected at the end of the experiment (day 28). Coupons inserts were fixed overnight with 5% Glutaraldehyde (Glutaraldehyde, 25% aqueous solution, Thermo Fisher United Kingdom) to preserve them until further analysis. Following this, inserts were fixed in 2% aqueous osmium tetroxide for 1 h at room temperature. A series of ethanol dilutions in distilled water were used for dehydrating the inserts in 15-min steps as follows: 75%, 95%, two steps of 100% ethanol, and 100% over anhydrous copper sulphate. The inserts were immersed in a 50/50% (v/v) solution of absolute ethanol and hexamethyldisilazane for 30 min and then transferred to 100% hexamethyldisilazane for a further 30 min. Samples were mounted onto a pin-stub using a Leit-C sticky tab and Leit-C plast, gold coated using an Edwards S150B sputter coater and examined in using a Tescan Vega3 LMU SEM at The University of Sheffield (UoS).