Lsm 900 laser

Embryos were allowed to develop to the appropriate stage and then imaged live or fixed in Methanol at -20 °C for 2 h and gradually rehydrated with 75% methanol-25% MEMFA (10X: 1 M MOPS, 20 mM EGTA, 10 mM MgSO4, 38% Formaldehyde), 50% methanol-50% MEMFA, 25% methanol-75% MEMFA and 100% MEMFA for 10 min each. Embryos were then permeabilized in PBDT (1 × PBS + 0.5% Triton X-100 + 1% DMSO) for several hours at room temperature and blocked in PBDT + 10% donkey serum for 1 h at room temperature. Primary antibodies were then added (in block solution). Primary antibodies used were: β-tubulin (Hybridoma), γ-Tubulin (Abcam) and CEP19 (Proteintech). The embryos were incubated in the antibody solution overnight at 4 °C. The next day embryos were washed in PBDT and then incubated in secondary antibodies diluted in the blocking solution at room temperature for 1 h. Imaging was performed on a Zeiss LSM 900 laser scanning confocal microscope with Airyscan imaging system. Image processing and colocalization analysis was done using the Zeiss ZEN 2.3 software.

All images were acquired at pixel dimensions of 1024 × 1024 and are shown as the maximum intensity of the Z‐projections using an LSM 900 laser scanning confocal microscope (Zeiss). For the measurement of immunofluorescence intensity, images were captured with the same laser power and the mean intensity of the fluorescence signals was measured and normalized to the mean DAPI signal intensity. The data were analysed using ZEN 3.4 Blue (Zeiss) and ImageJ software (National Institutes of Health) under the same processing parameters.