Following fixation, embryos were rinsed 3 times in PBS at room temperature, blocked at room temperature for 1 h in BDPF (1% Bovine Serum Albumin (Sigma A2153), 1% DMSO (Merck, D4540), 5% Foetal Calf Serum (Fisher 11573397) in 1× PBS) and then incubated overnight at 4°C in BDPF with 1:200 Phalloidin 594 (Invitrogen A12381) [70 (link)]. Trunks were dissected and mounted in a 1:1 mix of VectaShield with DAPI and PBST. The left pL1 neuromast was identified by its positioning at somite 6/7 [71 (link)] and imaged using Nikon A1 inverted confocal with a 40× objective, 8× optical zoom and 0.225 μm step size. Quantifications and statistical analysis of cilia polarity were carried out as described in [27 (link)], samples were blinded prior to analysis [69 ].
Other organizations :
Centre for Life, Newcastle University, Genome Institute of Singapore, Agency for Science, Technology and Research, Institute of Molecular and Cell Biology, Taibah University, Université de Montpellier, Institute for Neurosciences of Montpellier, Inserm, Centre Hospitalier de Valence, Centre Hospitalier Intercommunal de Poissy, Centre Hospitalier Universitaire de Martinique, King Abdullah University of Science and Technology
Phalloidin 594 (Invitrogen A12381) at 1:200 dilution
dependent variables
Cilia polarity
control variables
Fixation
Rinsing in PBS at room temperature
Blocking in BDPF (1% Bovine Serum Albumin, 1% DMSO, 5% Foetal Calf Serum in 1× PBS) at room temperature for 1 h
Incubation in BDPF overnight at 4°C
Trunk dissection
Mounting in a 1:1 mix of VectaShield with DAPI and PBST
Identification of the left pL1 neuromast at somite 6/7
Imaging using Nikon A1 inverted confocal with a 40× objective, 8× optical zoom and 0.225 μm step size
controls
Positive control: Not specified
Negative control: Not specified
Annotations
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