Plan apochromat objective

NIH-3T3 cells (cell line collection, IIET PAS) were grown in DMEM (4.5 % of glucose) containing 10 % FBS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), l-Glutamine (2 mM), β-mercaptoethanol (9 µM). For immunofluorescence cells were grown to 90–100 % confluency on sterile coverslips placed in 24-well plate. Cells were cultured in serum-free medium for 24 h before fixation to promote cilia formation (Pitaval et al. 2013 (link)). Cells were fixed in 3.7 % formalin in PBS and incubated for 10 min at room temperature, permeabilized with 0.1 % Triton X-100 in PBS (10 min), washed with PBS and blocked for 1 h with PBS containing 1 % FBS. Cells were then incubated for 1 h at RT with primary (Ab285: 1/100; mouse monoclonal against acetylated α-tubulin: 2 μg/ml) and secondary (Cy2 conjugated donkey anti-rabbit: 15 µg/ml, Cy5 conjugated goat anti-mouse: 15 µg/ml) antibodies, respectively. Control reactions were carried out without primary antibodies. Labeled cells were mounted in moviol based mounting medium containing DAPI (2 μg/ml) as a nuclei marker. Cells were imaged by spinning disc confocal microscope Zeiss Cell Observer SD equipped with Yokogawa CSU-X1A 5000 unit with the use of 63 × oil-immersed Plan-Apochromat objective (NA 1.4). Z-stacks across entire cells were acquired at Z increments of 0.4 μm. Images were processed with the use of ImageJ (NIH).

Live imaging of Nbs and pupae was performed at room temperature with either an Axio-observer inverted microscope (Carl Zeiss) equipped with a ×100 oil Plan-Apochromat objective lens (N/A 1.4), a spinning disk (CSUX-A1, Yokogawa), and an EMCCD Evolve camera (Princeton Instrument, Roper Scientific) or a Ti-DH inverted microscope (Nikon) equipped with a ×100 oil Plan-Apochromat objective lens (N/A 1.45), a spinning disk (CSU-W1-T1, Yokogawa), and a sCMOS camera (Roper Scientific). Images were acquired with Metamorph software (Molecular Device). Twelve to 20 Z of 0.5 µm steps were acquired every 20 s except in Fig. 3a–f, where one z plane was acquired every second (a-e) or every 5 sec (f). Fixed preparations and live embryos were observed using a DMI6000 inverted microscope equipped with a Plan-Apochromat ×63 objective (N/A 1.4) and a Leica TCS SP8 laser confocal imaging system. To induce the photoconversion of Sqh::Dendra2, cells were irradiated with a 405 nm laser (2.5% power, 1000 repetition) on a user-defined region for about 6–8 s using an Ilas2 system (Roper Scientific). Images in all figures are maximum projections except in Fig. 3a, b, d, e, f and DIC images.

Flowers at stage 13 and 15 were emasculated and pollinated on plants with mature pollen from the pACT11::RFP line. Immediately after pollination, stigmas were mounted between two coverslips maintained separated by four grease plugs placed at each coverslip corner. To maintain a constant humidity without adding liquid directly on the stigma surface, we used a wet piece of tissue in contact with the base of the stigma. Pollinated stigmas were observed under a Zeiss microscope (AxioObserver Z1) equipped with a spinning disk module (CSU-W1-T3, Yokogawa) using a 40x Plan-Apochromat objective (numerical aperture 1.1, water immersion). Serial confocal images were acquired in the entire volume of the stigma every 1 µm and every minute. Images were processed with Image J software and pollen tube lengths were measured.