3.5@ DAPI, Hoechst33342, and PI Staining of protonemata

Yuji Hiwatashi

 

[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

 

 

 

B. Hoechst33342 staining

[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

 

 

 

 

C. Propidium iodide staining

[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.

 
[Solution required]

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.