13 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
191
0.075% BRIJ, each for 3 mins, sections are reacted with 50
times diluted HRP-conjugated swine anti-rabbit antibody
for 30 mins at room temperature.
Note: Washing buffer containing BRIJ has a stronger effect, leading to a low background. Tween 20 instead of BRIJ can also be
used.
5. Visualization: After washing four times with PBS containing
0.075% BRIJ, each for 3 mins, sections are reacted 0.5 mg/ml of
3,3' -diaminobenzidine tetrahydrochloride with H20 2• The
duration of the reaction time in this step depends on the degree of color development (usually from 3 to 30 mins). After
washing under running tap water, sections are stained with
methyl green or hematoxyline-eosin, if nuclear staining is necessary. After treatment of the sections with serial concentrations of ethanol and with xylene, sections are mounted.
Note: If alkaline phosphatase (ALP) -conjugated antibody is
used, DIG nucleic acid detection kit (Boehringer) is useful.
The signal is dark green-gray in color. This system is very sensitive.
• Buffer 1: maleic acid, 0.1 mo1!l; NaCI, 0.15 molll; pH 7.5
Required solutions for
• Blocking solution: Blocking reagent (vial 6) is dissolved with detection with
10% (w/v) solution and stored at 4°C or -20°e.
ALP-conjuga• Buffer 2: Blocking solution is diluted to 1 % with Buffer 1.
ted antibody
• Washing buffer: 0.3% Tween-20 in Buffer 1.
• Buffer 3: Tris-HCI, 100 mmolll; NaCI, 100 mmolll; MgCh, 50
mmol/l; pH 9.5.
• Buffer 4: Tris-HCl, 10 mmolll; EDTA, 1 mmolll; pH 8.0
• Color substrate solution (freshly prepared before use): Add
NBT -solution (vial 4) and 35 I.tI X-phosphate-solution (vial
5) into 10 ml of Buffer 3. When blocking of endogenous alkali
phosphatase is necessary, 10 drops of levamisole (Dako
X3021) are added to the color substrate solution.
191
0.075% BRIJ, each for 3 mins, sections are reacted with 50
times diluted HRP-conjugated swine anti-rabbit antibody
for 30 mins at room temperature.
Note: Washing buffer containing BRIJ has a stronger effect, leading to a low background. Tween 20 instead of BRIJ can also be
used.
5. Visualization: After washing four times with PBS containing
0.075% BRIJ, each for 3 mins, sections are reacted 0.5 mg/ml of
3,3' -diaminobenzidine tetrahydrochloride with H20 2• The
duration of the reaction time in this step depends on the degree of color development (usually from 3 to 30 mins). After
washing under running tap water, sections are stained with
methyl green or hematoxyline-eosin, if nuclear staining is necessary. After treatment of the sections with serial concentrations of ethanol and with xylene, sections are mounted.
Note: If alkaline phosphatase (ALP) -conjugated antibody is
used, DIG nucleic acid detection kit (Boehringer) is useful.
The signal is dark green-gray in color. This system is very sensitive.
• Buffer 1: maleic acid, 0.1 mo1!l; NaCI, 0.15 molll; pH 7.5
Required solutions for
• Blocking solution: Blocking reagent (vial 6) is dissolved with detection with
10% (w/v) solution and stored at 4°C or -20°e.
ALP-conjuga• Buffer 2: Blocking solution is diluted to 1 % with Buffer 1.
ted antibody
• Washing buffer: 0.3% Tween-20 in Buffer 1.
• Buffer 3: Tris-HCI, 100 mmolll; NaCI, 100 mmolll; MgCh, 50
mmol/l; pH 9.5.
• Buffer 4: Tris-HCl, 10 mmolll; EDTA, 1 mmolll; pH 8.0
• Color substrate solution (freshly prepared before use): Add
NBT -solution (vial 4) and 35 I.tI X-phosphate-solution (vial
5) into 10 ml of Buffer 3. When blocking of endogenous alkali
phosphatase is necessary, 10 drops of levamisole (Dako
X3021) are added to the color substrate solution.