3.5@
DAPI, Hoechst33342, and PI Staining
of protonemata
[Introduction]
DAPI
(4',6-diamidino-2-phenylindole) emits blue fluorescence upon binding DNA and
can be excited with a mercury-arc lamp or with the UV lines of the argon-ion
laser. The blue-fluorescent DAPI associates with the minor groove of double
strand DNA, preferentially binding to AT clusters; there is a report that DAPI
binds to DNA sequences that contain as few as two consecutive AT base pairs,
perhaps employing a different binding mode. DAPI is readily soluble in water
but has limited solubility in phosphate-buffered saline (PBS). In P. patens, DAPI is impermeant into
living cells except sperms, and is only used for fixed cells.
@@@@@@@@ The
bisbenzimide dyes, Hoechst 33342 and Hoechst33258 are cell membrane-permeant,
although they are not so permeable in living P. patens cells. These dyes bind to DNA minor grooves and emit bright
blue fluorescence with UV light. Both dyes are quite soluble in water and up to
2% solutions can be prepared. These are relatively nontoxic. Hoechst 33342 has
slightly higher membrane-permeability than Hoechst 33258. These Hoechst dyes,
which can be excited with the UV spectral lines of the argon-ion laser or with
other conventional fluorescence excitation sources, exhibit relatively large
Stokes shifts (excitation/emission maxima ~350/460 nm), making them suitable
for multicolor labeling. The Hoechst 33258 and Hoechst 33342 dyes have
pH-dependent spectra when they do not bind to nucleic acids, with a much higher
fluorescence quantum yield at pH 5 than at pH 8.
@@@@@@@@ PI (Propidium
Iodide) is soluble in water and is not cell-permeant. PI is excited with
mercury- or xenon-arc lamps or with the argon-ion laser. PI binds to RNA as
well as DNA.
A.
DAPI staining (Murata and Sugai, 2000)
[Procedure]
1. Put the
sample in 10 ml of fixation solution and fix the sample for 10~60 min at room
temperature (or 4˚C overnight).
2. Discard
the fixation solution and incubate the sample in Tx-100 solution at 4˚C
overnight.
3. Discard
the Tx-100 solution and soak the sample in DAPI staining buffer for 10 min at
room temperature.
4. Discard
the DAPI staining buffer and add DAPI working solution in the sample.@ Incubate the sample for 10 min.
5. Wash the
sample with DAPI staining buffer twice.
6. Mount the
sample on a slide-glass with DAPI staining buffer. The sample can be observed with
epifluorescence microscopy, using an UV filter (excitation 365 nm). The stained
nucleus has bright blue fluorescence.
NOTE
- Samples stained with DAPI should be kept in dark, as DAPI is light sensitive
and the fluorescence fades quickly under light.
@[Solution required]
1.
Fixation solution
Stock solution |
10 ml |
Final conc. |
Formalin |
1 ml |
10% |
50 mM PIPES pH6.8 |
9 ml |
|
2.
Tx-100 solution
Stock solution |
10 ml |
Final conc. |
10% Triton X-100 |
1 ml |
1% |
DAPI staining buffer |
9 ml |
|
3.
DAPI staining buffer
Stock solution |
10 ml |
Final conc. |
1 M Tris pH8.0 |
0.1 ml |
10 mM |
0.5 M EDTA pH8.0 |
20 µl |
1 mM |
5 M NaCl |
0.3 ml |
150 mM |
H2O |
10 ml |
|
4.
DAPI working solution
Stock solution |
10 ml |
Final conc. |
DAPI |
1 µg |
0.1 µg |
DAPI staining buffer |
10 ml |
|
[Procedure]
1. Add the
sample in Hoechst33342 working solution and incubate the sample for 10 min.
2. Mount the
sample on a slide glass.
[Solution
required]
1.
1 mg/ml Hoechst33342
Stock solution |
1 ml |
Final conc. |
Hoechst33342 (4˚C, wako, ICN) |
1 mg |
1 mg/ml |
H2O |
1 ml |
|
Store
at 4˚C and use in 2 weeks.@
2.
10% TritonX-100
3. BCD liquid
medium
4. Hoechst33342
working solution
Stock solution |
1 ml |
Final conc. |
1 mg/ml Hoechst33342 |
5 µl |
5 µg/ml |
10% TritonX-100 |
10
µl |
0.05% |
BCD medium |
1 ml |
|
[Procedure]
1. Put the
sample in fixation solution and incubate the sample for one hour at room
temperature.
2. Wash the
sample with PBS three times.
3. Add RNase
working solution to the sample and incubate for 30 min at 37˚C.
4. Wash the
sample with PBS three times.
5. Add PI
solution to the sample and incubate for 10 min at 37˚C or overnight at
4˚C.
6. Wash the
sample with PBS three times.
7. Mount the
sample on a slide glass.
1.
Fixation solution
Stock solution |
10 ml |
Final conc. |
Formalin |
1 ml |
10% (v/v) |
10% NP-40 |
10 µl |
0.01% (v/v) |
PBS |
9 ml |
|
2.
PBS
@
3.
RNase A working solution
Stock solution |
10 ml |
Final conc. |
10 mg/ml RNase A |
0.1 ml |
100 µg/ml |
PBS |
9 ml |
|
3.
PI solution
Stock solution |
10 ml |
Final conc. |
Propidium iodide |
10 µg |
@1 µg/ml |
10 mg/ml RNase A |
10
µl |
10 µg/ml |
10% Tween-20 |
20
µl |
0.02% (v/v) |
PBS |
10 ml |
|
[References]
Murata,
T. and Sugai, M. (2000). Photoregulation of asymmetric cell division
followed by rhizoid development in the fern Ceratopteris prothalli. Plant Cell Physiol 41, 1313-20.