Dp52 camera

Flies were raised on standard Drosophila cornmeal–yeast–agar medium at 18°C and 25°C. The Oregon R stock from the Bloomington Drosophila Stock Center was used as wild-type control. The SuUR^ES mutation was first described by Belyaeva and the co-workers [83 (link)].
For acetic orcein staining of polytene chromosomes, glands were dissected in PBS (137 mM NaCl, 3 mM KCl, 8 mM NaH₂PO₄ and 2 mM KH₂PO₄), transferred to aceto-orcein solution (1% orcein in 45% acetic acid) for 10–15 min then to 55% lactic acid for 1–2 min and squashed. Phase-contrast images were captured with an Olympus BX51 microscope using a DP52 camera with a 100× oil immersion objective lens.

Polytene chromosomes were stained with aceto–orcein by standard methods [75 (link)]. Briefly, the salivary glands were dissected in 1× PBS, transferred to an aceto–orcein solution (1% orcein in 45% acetic acid) for 10–15 min incubation, followed by incubation in 55% lactic acid for 1–2 min and then were squashed. Phase contrast and fluorescent images were acquired using an Olympus BX51 microscope equipped with a 100×/1.30 Uplan FI Ph3 oil objective and a DP52 camera.
For ultrastructural analysis of polytene chromosomes, the same chromosome fixation protocol was employed as for immunostaining (see above). To analyze replication and the ultrastructure of chromosomes beyond the classic Abbe–Rayleigh limit of ~250 nm, three-dimensional (3D) structured illumination microscopy (3D-SIM) was applied using a Zeiss Elyra PS.1 microscopy system equipped with a Plan-Apochromat 63x/1.4 oil objective and the ZEN Black software (Carl Zeiss GmbH). Image stacks were obtained separately for each fluorochrome using 561 and 405 nm laser lines for excitation with appropriate emission filters [76 ].

Polytene chromosomes were fixed and stained with aceto-orcein by the method of Zhimulev et al. [39 (link)]. Phase contrast images were acquired with an Olympus BX51 microscope using a 100×/1.30 Uplan FI objective and a DP52 camera (Olympus, Tokyo, Japan). The same fixation procedure was used to prepare polytene chromosomes for three-dimensional, structured illumination microscopy (3D-SIM), which was performed with a Zeiss Elyra PS.1 microscope system using a Plan-Apochromat 63×/1.4 oil objective and the ZENBlack 2.1 software (Carl Zeiss GmbH, Jena, Germany). Image stacks for each fluorochrome were generated with 561, 488 and 405 nm laser excitation and appropriate emission filters [40 (link)].