4. The output of this script is an excel sheet displaying characteristics for each nucleus, such as the dimensions of the nuclei
(area, radii length, volume. . .) loci coordinates, loci-nuclear
envelope distances, loci to loci distances. . . The script can be
provided upon request.
Following this protocol (see Note 69), you will also be able to
perform super-resolution 3D-dSTORM acquisitions on your samples to study sub-resolution gene architecture evolution during
EMT/MET processes. However, the aim of this chapter is not to
develop super-resolution 3D-dSTORM imaging technique, please
refer to the relevant references [18] (see Note 70).
4 Notes
1. Non-exhaustive list: Sodium orthovanadate, TEMED, APS,
Acrylamide:Bis-Acrylamide, paraformaldehyde, formamide.
2. Supplementation with MEGS and hEGF brings a final concentration of 0.4% (v/v) of Bovine Pituitary Extract, 0.5 μg/mL
hydrocortisone, 10 ng/mL of human EGF, and 1 μg/mL of
recombinant human insulin-like growth factor I.
3. We use high precision coverslips (Marienfeld, reference
0107052) in order to lower optical aberrations due to irregularities on the surface of coverslips. Normal coverslips can also
be used.
4. This reaction is exothermic. Since pH is temperaturedependent, make sure to measure the pH when the solution
is at 25
C, and with a Tris-compatible electrode (with a double
junction). For 100 mL 1 M Tris–HCl pH 8.0, you will need
approximately 4.2 mL of 37% HCl to reach the desired pH.
For 100 mL 3 M Tris–HCl pH 6.8, you will need approximately 24 mL of 37% HCl to reach the desired pH.
For 500 mL 3 M Tris–HCl pH 8.8, you will need approximately 30 mL of 37% HCl to reach the desired pH.
For 100 mL 1 M Tris–HCl pH 7.5, you will need approximately 6.5 mL of 37% HCl to reach the desired pH. For more
precision, pH can also be adjusted using diluted HCl.
For 100 mL 0.5 M Tris–HCl pH 8.5, you will need
approximately 1.2 mL of 37% HCl to reach the desired
pH. For more precision, pH can also be adjusted using
diluted HCl.
5. Add protease inhibitors freshly before use.
6. Due to its smell and toxicity, manipulating β-mercaptoethanol
under a fume hood is highly recommended.
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Noe ´ mie Kempf et al.
(area, radii length, volume. . .) loci coordinates, loci-nuclear
envelope distances, loci to loci distances. . . The script can be
provided upon request.
Following this protocol (see Note 69), you will also be able to
perform super-resolution 3D-dSTORM acquisitions on your samples to study sub-resolution gene architecture evolution during
EMT/MET processes. However, the aim of this chapter is not to
develop super-resolution 3D-dSTORM imaging technique, please
refer to the relevant references [18] (see Note 70).
4 Notes
1. Non-exhaustive list: Sodium orthovanadate, TEMED, APS,
Acrylamide:Bis-Acrylamide, paraformaldehyde, formamide.
2. Supplementation with MEGS and hEGF brings a final concentration of 0.4% (v/v) of Bovine Pituitary Extract, 0.5 μg/mL
hydrocortisone, 10 ng/mL of human EGF, and 1 μg/mL of
recombinant human insulin-like growth factor I.
3. We use high precision coverslips (Marienfeld, reference
0107052) in order to lower optical aberrations due to irregularities on the surface of coverslips. Normal coverslips can also
be used.
4. This reaction is exothermic. Since pH is temperaturedependent, make sure to measure the pH when the solution
is at 25
C, and with a Tris-compatible electrode (with a double
junction). For 100 mL 1 M Tris–HCl pH 8.0, you will need
approximately 4.2 mL of 37% HCl to reach the desired pH.
For 100 mL 3 M Tris–HCl pH 6.8, you will need approximately 24 mL of 37% HCl to reach the desired pH.
For 500 mL 3 M Tris–HCl pH 8.8, you will need approximately 30 mL of 37% HCl to reach the desired pH.
For 100 mL 1 M Tris–HCl pH 7.5, you will need approximately 6.5 mL of 37% HCl to reach the desired pH. For more
precision, pH can also be adjusted using diluted HCl.
For 100 mL 0.5 M Tris–HCl pH 8.5, you will need
approximately 1.2 mL of 37% HCl to reach the desired
pH. For more precision, pH can also be adjusted using
diluted HCl.
5. Add protease inhibitors freshly before use.
6. Due to its smell and toxicity, manipulating β-mercaptoethanol
under a fume hood is highly recommended.
376
Noe ´ mie Kempf et al.