3.7@ GUS staining

Yuji Hiwatashi

 

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]

If you do not prefer to fix tissue, go to step 5. We usually do not fix tissue, which does not cause any problems.

 

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).

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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.

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2) wash solution

50 mM NaH2PO4 (pH 7.0)

Store at 4˚C.

 

3) X-Gluc substrate solution

 

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

 

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#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.

 

Procedure

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