3.7@ GUS staining
Introduction
GUS staining is a convenient way to examine the tissue- and stage-specific expression of reporter genes.@
The soluble X-Gluc product usually gives a broad
signal. If you prefer to determine distribution of GUS activity at high
resolution, use a low concentration (0.01%) of Triton X-100.@
1.
Fixation and Incubation with X-Gluc
[procedure]
1)
Add 200 µl fixation solution in wells of 96 well microtitreplate or
microtubes.
2) Put each sample (protonema, gametophore) in
each well of the microtitreplate or microtube.
3) Fix for 30 min at room temperature.
4) Wash 3 times with 50 mM NaH2PO4
(pH 7.0).
@
5) Add 100 µl of X-Gluc substrate solution into the wells of
microtitreplate or microtubes.
6) Apply a vacuum for 30 min in darkness.
7) Incubate for 24-48 hr at 37˚C in darkness. Cover the titerplate
with a plastic sheet (e.g. SaranWrap) in order to prevent the solution from
drying.
[solution
required]
1)
fixation solution
Stock solution |
/20 ml |
final concentration |
1% ( = 51 mM) MES (pH5.6) |
4 ml |
0.2% (10 mM) |
Formalin |
0.06 ml |
0.3% |
Mannitol |
1.09 g |
0.3 M |
H2O |
approx.15 ml (up to 20 ml) |
|
1% MES (adjust to pH5.6 with 0.1 M KOH) should
be stored at 4˚C.
@
2)
wash solution
50 mM NaH2PO4 (pH 7.0)
Store at 4˚C.
Stock solution |
/5 ml |
final concentration |
20 mg/ml X-Gluc/DMF #1 |
65 µl |
0.5 mM |
12.5 mM K3Fe(CN)6
#2 |
200 µl |
0.5 mM |
12.5 mM K4Fe(CN)6
#3 |
200 µl |
0.5 mM |
10% Triton X-100 |
5 – 25 µl |
0.01-0.05% |
50 mM NaH2PO4
(pH7.0) |
4.52 ml |
|
@
#1@ 20 mg X-Gluc dissolved in 1 ml N, N-dimethylformamide (=38.3 mM
X-Gluc)
#2@ 61.7 mg K3Fe(CN)6 dissolved in 15 ml H2O
#3@ 79.2 mg K4Fe(CN)6Ľ3H2O dissolved in 15 ml H2O
X-Gluc/DMF solution should be stored at –20˚C in darkness
K3Fe(CN)6 and K4Fe(CN)6
solutions should be filtered with 0.22 µm membrane filter and stored at
4˚C in dark.
Triton X-100 should be stored at 4˚C.
[Keys]
1) If the GUS activity appears weak, stain the tissue without fixation.
2) If samples are partially stained, decrease the amount of sample in a
well.
2.Fixation
and Dehydration
Chlorophyll interferes with observation of the
blue GUS stain. Dehydration using ethanol removes chlorophyll.
1) Sample (protonema, gametophore) after
incubation in X-Gluc.
2) Remove the substrate solution and add 200 µl of 5% formalin for
10 min into the sample for fixation.
3) Remove the formalin solution and soak the sample in 200 µl of
5% (v/v) acetic acid for 10 min.
4) Remove the acetic acid solution and soak the sample in 200 µl
of 30% (v/v) ethanol and dehydrate for 5 min.
5) Remove the ethanol solution and soak the sample in 200 µl of
50% (v/v) ethanol and dehydrate for 5 min.
6) Remove the ethanol solution and soak the sample in 200 µl of
70% (v/v) ethanol and dehydrated for 5 min.
7) Remove the ethanol solution and soak the sample in 200 µl of
100% ethanol and dehydrated for 5 min.
8) Remove the ethanol solution and soak the sample in 200 µl of
100% ethanol and dehydrate overnight at 4˚C.
9) Rinse with 200 µl of 100% ethanol.
10) Observe
Solution
required
1) 5% formalin
Stock solution |
/10 ml |
Final concentration |
Formalin |
0.5 ml |
5% |
H2O |
9.5 ml |
|
2) 5% acetic acid
Stock solution |
/10 ml |
Final concentration |
acetic acid |
0.5 ml |
5% |
H2O |
9.5 ml |
|
3) Ethanol series
30%, 50%, 70% ethanol/H2O, 100% ethanol