3 Methods
3.1 Specimen
Preparation
Procedure (adapted from a Leica protocol):
1. Seed cells in glass-bottom Petri dishes d ¼ 35 mm (CELLView,
Greiner Bio-One) at a density of ~100 cells/mm
2
.
2. Aspirate the cell culture medium.
3. Wash briefly 1Â with PBS.
4. Fix with 4% PFA in PBS for 20 min.
5. Permeabilize with PBS/Tx twice for 10 min.
6. Incubate with primary antibody overnight at 4
C (see Notes 1
and 2).
7. Wash 3Â with PBS/Tx over 2 h each time.
8. Incubate with secondary antibody for 2 h at RT.
9. Wash 3Â with PBS/Tx over 2 h each time.
10. Postfix with 4% PFA in PBS for 10 min (see Note 3).
11. Wash briefly 3Â with PBS.
12. Store at 4
C in PBS until mounting.
13. Before imaging, replace PBS with an appropriate mounting
medium.
The mounting medium for dSTORM (also known as the imaging buffer) has to be chosen based on the following criteria:
1. Fluorophores used. Alexa-647 works best in Gloxy or
Vectashield-containing media, while OxEA allows to use a
wider range of dyes, e.g., for multi-color imaging.
Vectashield-containing media are usually unsuitable for the
detection of light with wavelengths ≲550 nm and for excitation with wavelengths ≲500 nm due to autofluorescence.
2. Depth of the region of interest (ROI) inside the sample. A ROI
close to the coverslip (<200 nm) can be imaged with total
internal reflection fluorescence microscopy (TIRF) which
allows to reduce background fluorescence. This needs a
mounting medium with a refractive index significantly lower
than that of glass. Therefore, usually water-based media are
used (n % 1.33), e.g., Gloxy or OxEA. These media, however,
are less suitable for deeper (more than several μm from the
coverslip) imaging because of spherical aberrations due to the
refraction index mismatch. For such situations, buffers with a
refractive index close to that of glass (n % 1.52) are better
suited, e.g., Vectashield/TDE. Using highly inclined and
laminated optical sheet (HiLo) illumination, the sample is
excited with a light beam under an angle smaller than the
critical angle for the given coverglass/specimen interface that
274
Leonid Andronov et al.
3.1 Specimen
Preparation
Procedure (adapted from a Leica protocol):
1. Seed cells in glass-bottom Petri dishes d ¼ 35 mm (CELLView,
Greiner Bio-One) at a density of ~100 cells/mm
2
.
2. Aspirate the cell culture medium.
3. Wash briefly 1Â with PBS.
4. Fix with 4% PFA in PBS for 20 min.
5. Permeabilize with PBS/Tx twice for 10 min.
6. Incubate with primary antibody overnight at 4
C (see Notes 1
and 2).
7. Wash 3Â with PBS/Tx over 2 h each time.
8. Incubate with secondary antibody for 2 h at RT.
9. Wash 3Â with PBS/Tx over 2 h each time.
10. Postfix with 4% PFA in PBS for 10 min (see Note 3).
11. Wash briefly 3Â with PBS.
12. Store at 4
C in PBS until mounting.
13. Before imaging, replace PBS with an appropriate mounting
medium.
The mounting medium for dSTORM (also known as the imaging buffer) has to be chosen based on the following criteria:
1. Fluorophores used. Alexa-647 works best in Gloxy or
Vectashield-containing media, while OxEA allows to use a
wider range of dyes, e.g., for multi-color imaging.
Vectashield-containing media are usually unsuitable for the
detection of light with wavelengths ≲550 nm and for excitation with wavelengths ≲500 nm due to autofluorescence.
2. Depth of the region of interest (ROI) inside the sample. A ROI
close to the coverslip (<200 nm) can be imaged with total
internal reflection fluorescence microscopy (TIRF) which
allows to reduce background fluorescence. This needs a
mounting medium with a refractive index significantly lower
than that of glass. Therefore, usually water-based media are
used (n % 1.33), e.g., Gloxy or OxEA. These media, however,
are less suitable for deeper (more than several μm from the
coverslip) imaging because of spherical aberrations due to the
refraction index mismatch. For such situations, buffers with a
refractive index close to that of glass (n % 1.52) are better
suited, e.g., Vectashield/TDE. Using highly inclined and
laminated optical sheet (HiLo) illumination, the sample is
excited with a light beam under an angle smaller than the
critical angle for the given coverglass/specimen interface that
274
Leonid Andronov et al.