3.9@ 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 well 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 add Tx-100 solution into the sample 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 soon 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. 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 |
|
3. Tx-100
solution
Stock
solution |
10 ml |
Final conc. |
10% Triton
X-100 |
1 ml |
1% |
DAPI
staining buffer |
9 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 hoechest33342 working solution and incubate the sample for 10 min.
2. Mount the
sample on a slide glass.
[Solution
required]
1. 1 mg/ml
hoechest33342
Stock
solution |
1 ml |
Final conc. |
hoechest33342
(4˚C, wako, ICN) |
1 mg |
1 mg/ml |
H2O |
1 ml |
|
Store at
4˚C and use in 2 weeks.@
3. 10%
TritonX-100
4. BCD liquid
medium
5. Hoechest33342
working solution
Stock
solution |
1 ml |
Final conc. |
1 mg/ml
hoechest33342 |
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.