Vectashield plus dapi

Hemi-thoraces of adult Drosophila were prepared and stained as described (Weitkunat and Schnorrer 2014 (link)). Rabbit anti-Salm was used at 1:50 (Kühnlein et al. 1994 (link)), mouse anti-Flag (Sigma), mouse anti-V5 (Abcam), and rhodamine phalloidin (Invitrogen) were all used at 1:500. Nuclei were visualized by embedding in Vectashield plus DAPI (Vector Laboratories) and images were acquired on a Zeiss LSM780 confocal and processed with FIJI and Photoshop.

Tissues were fixed in 10% neutral buffered formalin overnight at room temperature. If necessary, tissues were decalcified in 14% EDTA pH 8.0 prior cryopreservation in a 30% sucrose gradient, then embedded in optimal cutting temperature compound (OCT) on dry ice. Frozen sections from adult bones were collected on a CryoJane tape system and mounted with Vectashield plus DAPI (Vector Labs).

In vitro cultured (microinjected) embryos/oocytes were fixed (at required developmental stages) with 4% para-formaldehyde, immuno-fluorescently (IF) stained and imaged in complete z-series by confocal microscopy (using FV10i confocal microscope, Olympus) as previously described (Mihajlovic et al., 2015 ); Supplementary Table SM5 summarizes the identity and combinations (plus employed concentrations) of primary and fluorescently conjugated secondary antibodies used. The majority of IF stained embryos/oocytes were counterstained for DNA, using DAPI containing mounting solution (Vectashield plus DAPI, Vector Labs) and as indicated some embryo samples were also counterstained against filamentous-actin, using fluorescently-labeled rhodamine-conjugated phalloidin (ThermoFisher, R415 – described in Mihajlovic and Bruce, 2016 (link)).

Centromeric satellite DNA was synthesized by annealing and extending two DNA oligos using TaKaRa ExTaq (TaKaRa), followed by subcloning into pCR™II-TOPO®vector(Thermo). DNA probes were prepared by cutting and labeling the plasmid DNA with biotin, using the Nick Translation Kit (Roche). Medaka fibroblast cells were treated with 0.05 µg/ml of corcemid (for probe1,2) or 1 µM of nocodazole (for probe3, 4, all) for 4–5 h. After trypsinization, cells were hypotonically swollen in 75 mM KCl for 20 min, fixed with ice-cold Carnoy’s solution (1:3 acetic acid: methanol), then spread onto slides. After RNase treatment and denaturation of chromosomal DNA, hybridization was carried out by dropping probe DNA solution onto slides and incubating at 37 °C for overnight. After washing, chromosomal DNA was incubated with avidin-FITC (Vector Laboratories) for 1 h. After the final wash, slides were mounted with Vectashield Plus DAPI (Vector Laboratories). Images were acquired using a fluorescence microscope (LSM710; Zeiss).

Embryos were fixed in 4% PFA (20 minutes, 37 °C) and prepared for confocal-based immuno-fluorescence microscopy as follows (at room temperature in 150 μl, unless stated); i) three 5 minute PBS washes, ii) 20 minutes in 0.5% (in PBS) Triton X-100 (Fgfr2 immuno-staining 0.1% Triton X-100), iii) three 10 minute PBS-Tween 20 (0.15%; PBS-T) washes, iv) one 10 minute NH₄Cl (50 mM in PBS) wash, v) one 4 hour 3% BSA (in PBS-T; BSA-PBS-T) blocking-step (4 °C), vi) overnight primary antibody (diluted in BSA-PBS-T) incubation (4 °C), vii) three 10 minute PBS-T washes, viii) 4 hour BSA-PBS-T blocking-step (4 °C), ix) 1 hour fluorescently-labelled secondary antibody incubation (diluted in BSA-PBS-T, 4 °C), x) three 10 minute PBS-T washes, xi) 30 minutes PBS wash, and xii) mounting (Vectashield plus DAPI, Vector Labs) on glass-bottomed, poly-L-lysine coated culture dishes. For phospho-Yap1 (pYap1), some embryos were pre-treated with 1000 units λ-phosphatase (sc-200312, Santa Cruz Biotech) according to manufacturers protocol. Primary and secondary antibody details and dilutions are given in Supplementary Table ST14. Note, that for embryos double immuno-stained for Nanog and Gata4, the rat monoclonal version of the anti-Nanog antibody was used. Embryos were imaged on IX81 upright or Fluoview Fv10i confocal microscopes (Olympus).