medium on a glass slide using a tweezer and cannulas (see
Note 21).
7. Analyze samples by fluorescence microscopy using either a
bright-field microscope or a confocal laser scanning microscope
(CLSM) using appropriate setting (see Note 23).
A staining example is shown in Fig. 3b, c, f, g using PLT1 DIGand PLT3 FITC-labeled probes, respectively. Whereas Fig. 3b, f
shows light microscopy images, Fig. 3c, g is acquired at a CLSM.
3.9.3 HRP-Based TSA
Staining
1. Wash the Sieves Twice with TNT Buffer.
2. Incubate the sieves for 5–15 min in fresh TSA staining solution
(see Note 24).
3. Wash the sieves four times with TNT buffer for 5 min each.
4. Wash the sieves four times with TNT buffer for 10 min each.
5. Transfer and separate the roots in a drop of DAPI-containing
anti-fading mounting medium on a glass slide using a tweezer
and cannulas (see Note 21).
6. Analyze samples using a CLSM. Microscope settings depend
on the fluorescent dye used (see Note 25).
A staining example is shown in Fig. 3d, h using PLT1 DIG- and
PLT3 FITC-labeled probes, respectively, each in combination with
a TSA-CY3 substrate.
3.10 Double-TSA
Labeling
1. Follow the experimental procedure described in Subheading
3.4 up to step 2 of Subheading 3.7.
2. At step 3 of Subheading 3.7, apply two different labeled probes
in hybridization mix to the tissue (e.g., one labeled with DIG,
the other labeled with FITC (see Note 26)) and incubate the
independent sieves 7–16 h at the selected hybridization
temperature.
3. Wash three times with pre-warmed washing solution 1, first for
10 min, then for 60 min, and finally for 20 min at hybridization
temperature.
4. Wash the sieves twice with washing solution 2 and twice with
washing solution 3 for 20 min each at room temperature,
to remove nonspecific and/or repetitive DNA/RNA
hybridization.
5. Wash the sieves three times in PBS-T for 10 min each.
6. Incubate the sieves in blocking solution for 60 min.
7. Add one antibody directed against one labeled probe diluted in
blocking solution to the well and incubate the sieves in the well
for 4–15 h at 21
C (see Note 27).
8. Wash the sieves eight times with PBS-T for 10 min each.
RNA-FISH
15
Note 21).
7. Analyze samples by fluorescence microscopy using either a
bright-field microscope or a confocal laser scanning microscope
(CLSM) using appropriate setting (see Note 23).
A staining example is shown in Fig. 3b, c, f, g using PLT1 DIGand PLT3 FITC-labeled probes, respectively. Whereas Fig. 3b, f
shows light microscopy images, Fig. 3c, g is acquired at a CLSM.
3.9.3 HRP-Based TSA
Staining
1. Wash the Sieves Twice with TNT Buffer.
2. Incubate the sieves for 5–15 min in fresh TSA staining solution
(see Note 24).
3. Wash the sieves four times with TNT buffer for 5 min each.
4. Wash the sieves four times with TNT buffer for 10 min each.
5. Transfer and separate the roots in a drop of DAPI-containing
anti-fading mounting medium on a glass slide using a tweezer
and cannulas (see Note 21).
6. Analyze samples using a CLSM. Microscope settings depend
on the fluorescent dye used (see Note 25).
A staining example is shown in Fig. 3d, h using PLT1 DIG- and
PLT3 FITC-labeled probes, respectively, each in combination with
a TSA-CY3 substrate.
3.10 Double-TSA
Labeling
1. Follow the experimental procedure described in Subheading
3.4 up to step 2 of Subheading 3.7.
2. At step 3 of Subheading 3.7, apply two different labeled probes
in hybridization mix to the tissue (e.g., one labeled with DIG,
the other labeled with FITC (see Note 26)) and incubate the
independent sieves 7–16 h at the selected hybridization
temperature.
3. Wash three times with pre-warmed washing solution 1, first for
10 min, then for 60 min, and finally for 20 min at hybridization
temperature.
4. Wash the sieves twice with washing solution 2 and twice with
washing solution 3 for 20 min each at room temperature,
to remove nonspecific and/or repetitive DNA/RNA
hybridization.
5. Wash the sieves three times in PBS-T for 10 min each.
6. Incubate the sieves in blocking solution for 60 min.
7. Add one antibody directed against one labeled probe diluted in
blocking solution to the well and incubate the sieves in the well
for 4–15 h at 21
C (see Note 27).
8. Wash the sieves eight times with PBS-T for 10 min each.
RNA-FISH
15