Zen black 2

Raw images were acquired on a Zeiss Axio Observer Z1 SR using ELYRA PS.1 structured illumination microscopy with a × 100/1.46 Plan Apochromatic objective and a PCO scientific CMOS camera. Four solid-state lasers (405, 488, 561 and 647 nm) were used to take 40–60 Z-slices at 116 nm interval and five grid rotations. Images were acquired and processed with Zeiss Zen Black 2.1 software. Channel alignment for chromatic aberration was performed using multispectral beads.

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)].

Cells were grown on microscope cover glasses coated with 2.5 µg/ml of Collagene R Solution (Serva, Heidelberg, Germany) and stained with 300 nM MitoTracker Deep Red FM (Invitrogen), followed by treatment with IZI1551, alone or in combinations with TL32711 and ABT-199 After treatment, cells were washed with PBS and fixed using 4% paraformaldehyde for 30 min. Cells were permeabilised with 0.1% Triton X-100 in PBS and blocked with 4% Bovine Serum Albumin (BSA) before incubation with 5 μg/ml of monoclonal cytochrome c antibody (Clone 6H2.B4; Thermo Fisher Scientific) in 2% BSA/PBS. After incubation at room temperature for 1 h, cells were washed three times with PBS and incubated with 4 μg/ml of Alexa Fluor 488-labeled secondary antibody (#A-11029, Thermo Fisher Scientific) for 45 min. After 3 PBS washes, cells were mounted (Fluoromount-G, SouthernBiotech). Images were captured using a confocal laser scanning microscope (LSM 710, Carl Zeiss) equipped with a Plan Apochromat ×63/1.40 DIC M27 (Carl Zeiss, Jena, Germany) oil-immersion objective. MitoTracker was excited with a 633 nm laser and its emission was detected from 638 to 759 nm. Alexa Fluor 488 was excited with a 488 nm laser and detected with a 496/602 emission filter. Secondary antibody controls did not provide signal above background noise. Image processing and analysis was performed with Zen black 2.1 software (Carl Zeiss).

NIPA (Sigma-Aldrich, St. Louis, MO, USA, HPA024023) antibody was used for immunofluorescence experiments. Alexa Fluor-488 conjugated anti-rabbit IgG was purchased from Life Technologies. Different ALCL cells were fixed on slides with paraformaldehyde and permeabilized with methanol. Cells were stained with primary antibodies for 2 h prior to incubation with fluorophore-conjugated secondary antibodies for 1 h at room temperature in the dark. Mounting of slides was performed using the Gold Antifade reagent (Invitrogen). Slides were viewed with a Zeiss LSM 880 microscope (63×/1.4 Plan-Apo oil objective). Images were processed with Zeiss Zen Black 2.1 software.

The Airyscan detector upgrade on a confocal laser scanning microscope Zeiss LSM 780 was run in super-resolution mode (63× plan apochromatic oil immersion objective, 32 GaAsP detectors) [32 (link)]. The excitation lasers and emission detection windows are listed in Supplementary Table 7. For each field of view, a z-stack was taken and after post-processing with default Airyscan filtering, a 3D reconstruction was performed with Zen Black 2.1 software (Version 13.0.0.0, Carl Zeiss, Germany) with automatic Airyscan filter strength. The final images were obtained by a maximum intensity projection of the z-stack. The histogram of signal intensities was adjusted to maximize visibility of gene probe-conferred positive signals and the same values were used as thresholds to evaluate negative controls of the respective experiment to estimate eventual false positives. For better representation, fluorescent signals were consistently pseudocoloured, not resembling their original labeling dye color.

An ELYRA PS.1 microscope (Carl Zeiss) with a 63× oil immersion objective was used for structured-illumination microscopy (SIM) with five phases and three to five rotations of the SIM grid and acquisition times of 100–150 ms. All channels were acquired independently and subsequently. Raw confocal SIM images were processed and generated using the ZEN Black 2.1 software (Carl Zeiss).

Immunostaining was performed as described here [22 (link)]. SOX9 primary antibodies (HPA001758, Merck KGaA, Darmstadt, Germany) were used in dilution 1:100 and the corresponding secondary antibodies in dilution 1:400 (711-165-152 Jackson ImmunoResearch Europe Ltd, Cambridge House, St. Thomas’ Place, UK ). Nuclei were stained with DAPI (D8417, Merck KGaA, Darmstadt, Germany) for 20 minutes in concentration 10 µg per ml. The samples were examined by confocal scanning laser microscopy. Images were processed with microscope software (ZEN Black 2.0, Zeiss AG, Oberkochen, Germany).