3.11@ GUS staining
Introduction
GUS staining is a convenient way to examine the tissue- and
stage-specific expression of reporter genes.@
Soluble
X-Gluc product usually gives a broad signal. If you prefer to determine distribution
of GUS activity in fine resolution, use 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 temp.
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) Vacuum for
30 min in darkness.
7) Incubate
for 24-48 hr at 37˚C in darkness. Cover the titerplate with a plastic seat
(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 up to 1 ml with N, N-dimethylformamide (=38.3 mM X-Gluc)
#2@ 61.7 mg K3Fe(CN)6 up to 15 ml with H2O
#3@ 79.2 mg K4Fe(CN)6Ľ3H2O up to 15 ml with
H2O
X-Gluc/DMF solution should be stored at –20˚C in
dark
K3Fe(CN)6
and K4Fe(CN)6 solutions should be filtrated 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 sample amount in a well.
2.Fixation
and Dehydration
Although
chlorophyll interferes to observe blue signal of GUS staining, 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
ethanol 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