GUS染色 - motosehiroyasu @ ウィキ

GUS staining (4 Nov. 2008)

• 90% (v/v) acetone pre-chilled at 4℃
• 200 mM NaH2PO4 (adjust pH at 7.0 by NaOH and autoclave)
• 25 mM K3Fe(CN)6 (Potassium ferricyanide)
• 25 mM K4Fe(CN)6 (Potassium ferrocyanide)
• 10% (v/v) Triton X-100
• 20 mg mL-1 X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide Cyclohexylammonium salt) in DMSO.
• GUS staining solution
  • 200 mM NaH2PO4 2500 µL
  • 25 mM K3Fe(CN)6 1000 µL
  • 25 mM K4Fe(CN)6 1000 µL
  • 10% Triton X-100 50 µL
  • 20 mg/mL X-Gluc 125 µL
  • H2O 325 µL
  • Total 5000 µL
• 70% ethanol
• Fixing solution (ethanol : acetic acid = 9 :1 volume)
• Clearing solution (chloral hydrate : glycerol : H2O = 8 g : 1 mL : 2 mL)
  • (Chloral hydrate is identical to trichloroacetaldehyde monohydrate.)

1. Incubate samples in 90% acetone for 15 min at 4℃.

 ○This step is required for staining of shoot organs (leaves, stems, etc).

2. Wash with 100 mM NaH2PO4 pH 7.0.
3. Incubate samples in the GUS solution.

 ○Vacuum for 5-10 min to infiltrate GUS solution into samples.
 ○Staining condition is dependent on samples.
    Ordinary for 1 h ~ overnight at room temperature.
    Incubate samples at 37℃ if GUS staining is obscure.

4. Wash with 70% ethanol.
5. Incubate in fixing solution (r. t. about 30 min).
6. Incubate in clearing solution (r. t. for 1 hour ~ overnight).