Cells and embryos were fixed in 4% PBS–paraformaldehyde (PFA)
(15710, Electron Microscopy Sciences) for 20 min at room temperature and
subsequently washed twice with 0.1% Tween–PBS. To stain centrosomes,
cells and embryos were fixed in ice-cold methanol for 5 min at 4 °C.
Peremeabilization was done in PBS containing 0.3% Triton X-100 and 0.1 M glycine
for 30 min at room temperature. Cells were incubated with primary antibodies
(Supplementary Table
2 ) at 4 °C overnight, followed by incubation with
fluorescently conjugated Alexa Fluor secondary antibodies (Thermo Fisher
Scientific) for 2 h at room temperature (Supplementary Table 2 ). Both primary and secondary
antibodies were diluted in 1% BSA, 0.1% Tween–PBS.
Images were acquired on an inverted SP5 confocal microscope (Leica
Microsystems) with a Leica HC PL APO 1.4 NA 63× oil objective or on an
inverted SP8 confocal microscope (Leica Microsystems) with a Leica HC PL APO CS2
1.4 NA 63× oil objective or a Leica Fluotar VISIR 0.95 NA 25 x water
objective.
(15710, Electron Microscopy Sciences) for 20 min at room temperature and
subsequently washed twice with 0.1% Tween–PBS. To stain centrosomes,
cells and embryos were fixed in ice-cold methanol for 5 min at 4 °C.
Peremeabilization was done in PBS containing 0.3% Triton X-100 and 0.1 M glycine
for 30 min at room temperature. Cells were incubated with primary antibodies
(
2
fluorescently conjugated Alexa Fluor secondary antibodies (Thermo Fisher
Scientific) for 2 h at room temperature (
antibodies were diluted in 1% BSA, 0.1% Tween–PBS.
Images were acquired on an inverted SP5 confocal microscope (Leica
Microsystems) with a Leica HC PL APO 1.4 NA 63× oil objective or on an
inverted SP8 confocal microscope (Leica Microsystems) with a Leica HC PL APO CS2
1.4 NA 63× oil objective or a Leica Fluotar VISIR 0.95 NA 25 x water
objective.