final concentration of 1 mg/mL to pre-hybridization mix. Add
labeled probe (see Note 19) and denature the solution for
10 min at 65
C followed by incubation for 2 min on ice.
3. Distribute the sieves into multi-well plates.
4. Incubate the independent sieves 7–16 h with hybridization mix
containing the individual probes at the selected hybridization
temperature.
5. 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.
6. 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.
3.8 Antibody Binding
1. Wash the sieves three times in PBS-T for 10 min each.
2. Incubate the sieves in blocking solution for 60 min.
3. Add the selected antibody diluted in blocking solution to the
well and incubate the sieves in the well for 4–15 h at 21
C (see
Note 20).
4. Wash the sieves eight times with PBS-T for 10 min each.
3.9 Staining
and Visualization
of RNA
For RNA visualization choose between the three staining methods
Subheadings 3.9.1–3.9.3 (see Note 20).
3.9.1 Chromogenic
AP-Based Staining Using
NBT/BCIP as Substrate
1. Wash the sieves twice with AP buffer.
2. Incubate the sieves in pre-warmed (37
C) AP staining solution
in the dark at 37
C until a dark blue indigo staining is visible
(initially check every 5 min; afterwards the periods can be
extended).
3. Stop the reaction with 100% (v/v) ethanol for 10 min.
4. Rehydrate tissues with 50% (v/v) ethanol for 10 min.
5. Rehydrate tissues two times with PBS-T for 10 min.
6. For DIC microscopy, the roots are transferred and separated in
a drop of clearing solution on a glass slide using a tweezer and
cannulas (see Note 21).
A staining example is shown in Fig. 3a, e using PLETHORA1
(PLT1) DIG- and PLETOHORA3 (PLT3) FITC-labeled probes.
3.9.2 Fluorescent
AP-Based Staining Using
Fast Red as a Substrate
1. Wash samples twice with AP buffer.
2. Incubate samples in pre-warmed (37
C) Fast Red staining
solution.
RNA-FISH
13
labeled probe (see Note 19) and denature the solution for
10 min at 65
C followed by incubation for 2 min on ice.
3. Distribute the sieves into multi-well plates.
4. Incubate the independent sieves 7–16 h with hybridization mix
containing the individual probes at the selected hybridization
temperature.
5. 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.
6. 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.
3.8 Antibody Binding
1. Wash the sieves three times in PBS-T for 10 min each.
2. Incubate the sieves in blocking solution for 60 min.
3. Add the selected antibody diluted in blocking solution to the
well and incubate the sieves in the well for 4–15 h at 21
C (see
Note 20).
4. Wash the sieves eight times with PBS-T for 10 min each.
3.9 Staining
and Visualization
of RNA
For RNA visualization choose between the three staining methods
Subheadings 3.9.1–3.9.3 (see Note 20).
3.9.1 Chromogenic
AP-Based Staining Using
NBT/BCIP as Substrate
1. Wash the sieves twice with AP buffer.
2. Incubate the sieves in pre-warmed (37
C) AP staining solution
in the dark at 37
C until a dark blue indigo staining is visible
(initially check every 5 min; afterwards the periods can be
extended).
3. Stop the reaction with 100% (v/v) ethanol for 10 min.
4. Rehydrate tissues with 50% (v/v) ethanol for 10 min.
5. Rehydrate tissues two times with PBS-T for 10 min.
6. For DIC microscopy, the roots are transferred and separated in
a drop of clearing solution on a glass slide using a tweezer and
cannulas (see Note 21).
A staining example is shown in Fig. 3a, e using PLETHORA1
(PLT1) DIG- and PLETOHORA3 (PLT3) FITC-labeled probes.
3.9.2 Fluorescent
AP-Based Staining Using
Fast Red as a Substrate
1. Wash samples twice with AP buffer.
2. Incubate samples in pre-warmed (37
C) Fast Red staining
solution.
RNA-FISH
13