Thunder imager 3d

Chromosome images were captured using a Zeiss Axio Scope A1 epifluorescence microscope fitted with a high-resolution microscopy camera AxioCam MRm Rev. 3 (Carl Zeiss Ltd. Oberkochen, Germany) and a Leica DM6 B (Leica microsystems, Macquarie park, Australia). Images were analysed using Metasystems Isis FISH Imaging System V 5.5.10 software (Metasystems, Altlussheim, Germany) as well as Thunder Imager 3D (Leica microsystems).

Cells were fixed in 4% PFA at room temperature for 30 min and washed with PBS twice. Cells were then treated with 10% FBS, 5% BSA and 0.2% Triton X-100 in PBS for 15 min, allowing blocking and permeabilization. Incubation with various primary antibodies was overnight, 4 °C. Following primary antibody incubation and washes in 1X PBS, the cells were incubated with the Alexa-Fluor 488 or 594 secondary antibodies (1:400, Molecular Probes) and nuclei were stained with Dapi (1:5000, Calbiochem). Cells were visualized with a 10×/0.30 or 20×/0.40 (dry lens) objective using inverted microscopes (DMI4000 or THUNDER Imager 3D, Leica Microsystems). The images were captured with a digital camera (DFC365 FX, Leica Microsystems) using LAS-AF software (Leica Microsystems).
Antibodies used in this study are as following: cTNT (MA5-12960, Thermo Fisher Scientific), GATA4 (SC-25310 Santa Cruz), α-MHC (MF20) (14-6503-82, eBioscience), DDR2 (sc-81707, Santa Cruz), CD31 (e-ab-30820, Elabscience), MLC2v (ab79935, Abcam), α-Actinin (A7811, Merck).