61. For DNA-FISH probes from fosmids, the covered genomic
region might be too narrow to be clearly visible by fluorescence. To overcome this issue you can combine 2 consecutives
fosmids to cover a larger region. In this case combine 10 to
15 ng of each fosmid (obtained during the nick translation
step) in the microtube.
62. Cot-1 human DNA is used to block nonspecific hybridization
to repetitive sequences in the mammalian genome and is therefore important to reduce background for microscopy.
63. Be careful as the pellet might detach from the tube wall.
64. During this step, avoid the exposure of the labeled DNA-FISH
probes to direct light.
65. In the case of multiple FISH labeling, premix all three
DNA-FISH probes in a microtube, vortex and pulse spin
before dropping the probe mixture on the glass slide.
66. As the cells grew on the upper side of the coverslip, do not
directly touch the fixed cells with the tissue.
67. Cover the entire junction between the coverslip and the slide to
hermetically seal the coverslip.
68. Should the coverslip stick to the slide, add ~100 μL of 2Â SSC
onto the coverslip to the slide junction and push very gently
the coverslip toward the edges of the slide to avoid sample
friction damages.
69. For super-resolution imaging, we advise you to use Alexa 647
dye and to skip the Hoechst staining step to avoid additional
background during the acquisition.
70. 3D-dSTORM acquisition was performed on an inverted
microscope equipped with a 100X TIRF (NA 1.49) objective,
containing a quadrichroic mirror (405 nm/488 nm/565 nm/
640 nm) for simultaneous laser excitation. Lasers are delivered
via an optical fiber with the following power and wavelengths:
100 mW at 405 nm and 300 mW at 647 nm. Fluorescence is
collected using single band pass emission filter. The microscope
is equipped with a motorized stage in z and a perfect focus
system to correct for drifts. A MicAO 3DSR (Imagine Optic)
was used to induce astigmatism to the PSF for acquisitions
in 3D.
We used the SAFe Reagent (Abbelight) as an optimized
buffer for dSTORM acquisitions.
References
1. Pastushenko I, Blanpain C (2019) EMT transition states during tumor progression and
metastasis. Trends Cell Biol 29:212–226.
https://doi.org/10.1016/J.TCB.2018.12.
001
2. Hao Y, Baker D, ten Dijke P et al (2019)
TGF-β-mediated epithelial-mesenchymal transition and cancer metastasis. Int J Mol Sci
20:2767.
https://doi.org/10.3390/
ijms20112767
382
Noe ´ mie Kempf et al.
region might be too narrow to be clearly visible by fluorescence. To overcome this issue you can combine 2 consecutives
fosmids to cover a larger region. In this case combine 10 to
15 ng of each fosmid (obtained during the nick translation
step) in the microtube.
62. Cot-1 human DNA is used to block nonspecific hybridization
to repetitive sequences in the mammalian genome and is therefore important to reduce background for microscopy.
63. Be careful as the pellet might detach from the tube wall.
64. During this step, avoid the exposure of the labeled DNA-FISH
probes to direct light.
65. In the case of multiple FISH labeling, premix all three
DNA-FISH probes in a microtube, vortex and pulse spin
before dropping the probe mixture on the glass slide.
66. As the cells grew on the upper side of the coverslip, do not
directly touch the fixed cells with the tissue.
67. Cover the entire junction between the coverslip and the slide to
hermetically seal the coverslip.
68. Should the coverslip stick to the slide, add ~100 μL of 2Â SSC
onto the coverslip to the slide junction and push very gently
the coverslip toward the edges of the slide to avoid sample
friction damages.
69. For super-resolution imaging, we advise you to use Alexa 647
dye and to skip the Hoechst staining step to avoid additional
background during the acquisition.
70. 3D-dSTORM acquisition was performed on an inverted
microscope equipped with a 100X TIRF (NA 1.49) objective,
containing a quadrichroic mirror (405 nm/488 nm/565 nm/
640 nm) for simultaneous laser excitation. Lasers are delivered
via an optical fiber with the following power and wavelengths:
100 mW at 405 nm and 300 mW at 647 nm. Fluorescence is
collected using single band pass emission filter. The microscope
is equipped with a motorized stage in z and a perfect focus
system to correct for drifts. A MicAO 3DSR (Imagine Optic)
was used to induce astigmatism to the PSF for acquisitions
in 3D.
We used the SAFe Reagent (Abbelight) as an optimized
buffer for dSTORM acquisitions.
References
1. Pastushenko I, Blanpain C (2019) EMT transition states during tumor progression and
metastasis. Trends Cell Biol 29:212–226.
https://doi.org/10.1016/J.TCB.2018.12.
001
2. Hao Y, Baker D, ten Dijke P et al (2019)
TGF-β-mediated epithelial-mesenchymal transition and cancer metastasis. Int J Mol Sci
20:2767.
https://doi.org/10.3390/
ijms20112767
382
Noe ´ mie Kempf et al.