9. Wash the sieves twice with TNT buffer.
10. Incubate the sieves for 5–15 min in fresh TSA staining
solution.
11. Wash the sieves four times with TNT buffer for 5 min each.
12. Deactivate the HRP by sieve incubation in 100 mM glycine
pH 2.0 and 0.1% (v/v) Tween
® 20 for 10 min.
13. Wash the sieves with PBS-T buffer.
14. Add the other antibody directed against the other labeled
probe diluted in blocking solution to the well and incubate
the sieves in the well for 4–15 h at 21
C.
15. Wash the sieves eight times with PBS-T for 10 min each.
16. Wash the sieves twice with TNT buffer.
17. Incubate the sieves for 5–15 min in fresh TSA staining solution
(use another fluorescent dye as in the first round) (see Note
25).
18. Wash the sieves four times with TNT buffer for 5 min each.
19. Wash the sieves four times with TNT buffer for 10 min each.
20. 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).
21. Analyze samples using a CLSM. Microscope settings depend
on the fluorescent dye used (see Note 25).
A double-TSA staining example is shown in Fig. 3i, using PLT1
DIG probes visualized by HRP and TSA CY3 as well as PLT3 FITC
probes stained by HRP and TSA FITC.
The combination of HRP- and AP-based staining is straightforward as both antibodies can be applied at the same time. The
TSA staining reaction should be performed (see Subheading 3.9.3,
steps 1–4) before the AP staining reaction (see Subheading 3.9.2,
steps 1–7).
3.11 Examples
for the Different
Staining Methods
Figure 3 shows the results of different staining methods using DIGor FITC-labeled probes for the detection of PLT mRNAs (Fig. 3).
The overlapping expression pattern of PLT1 and PLT3 genes is
demonstrated. Both genes are highly expressed in the region of the
quiescent center (QC) of the root and the surrounding stem cells,
which can be visualized with all three methods. Using light microscopy, NBT/BCIP staining shows a significant better contrast compared to Fast Red staining. However, Fast Red-stained tissue
provides a much higher cellular resolution than NBT/BCIP staining when observed by confocal microscopy, particularly when combined with DAPI counterstaining of the DNA. However, all
though method allows to identify and demonstrate a cell-specific
mRNA expression pattern, only the TSA system permits to
16
Thomas Dresselhaus and Andrea Bleckmann
10. Incubate the sieves for 5–15 min in fresh TSA staining
solution.
11. Wash the sieves four times with TNT buffer for 5 min each.
12. Deactivate the HRP by sieve incubation in 100 mM glycine
pH 2.0 and 0.1% (v/v) Tween
® 20 for 10 min.
13. Wash the sieves with PBS-T buffer.
14. Add the other antibody directed against the other labeled
probe diluted in blocking solution to the well and incubate
the sieves in the well for 4–15 h at 21
C.
15. Wash the sieves eight times with PBS-T for 10 min each.
16. Wash the sieves twice with TNT buffer.
17. Incubate the sieves for 5–15 min in fresh TSA staining solution
(use another fluorescent dye as in the first round) (see Note
25).
18. Wash the sieves four times with TNT buffer for 5 min each.
19. Wash the sieves four times with TNT buffer for 10 min each.
20. 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).
21. Analyze samples using a CLSM. Microscope settings depend
on the fluorescent dye used (see Note 25).
A double-TSA staining example is shown in Fig. 3i, using PLT1
DIG probes visualized by HRP and TSA CY3 as well as PLT3 FITC
probes stained by HRP and TSA FITC.
The combination of HRP- and AP-based staining is straightforward as both antibodies can be applied at the same time. The
TSA staining reaction should be performed (see Subheading 3.9.3,
steps 1–4) before the AP staining reaction (see Subheading 3.9.2,
steps 1–7).
3.11 Examples
for the Different
Staining Methods
Figure 3 shows the results of different staining methods using DIGor FITC-labeled probes for the detection of PLT mRNAs (Fig. 3).
The overlapping expression pattern of PLT1 and PLT3 genes is
demonstrated. Both genes are highly expressed in the region of the
quiescent center (QC) of the root and the surrounding stem cells,
which can be visualized with all three methods. Using light microscopy, NBT/BCIP staining shows a significant better contrast compared to Fast Red staining. However, Fast Red-stained tissue
provides a much higher cellular resolution than NBT/BCIP staining when observed by confocal microscopy, particularly when combined with DAPI counterstaining of the DNA. However, all
though method allows to identify and demonstrate a cell-specific
mRNA expression pattern, only the TSA system permits to
16
Thomas Dresselhaus and Andrea Bleckmann