412
HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
- Avidin-Cy5 (BDS), 1 mg/ml in sterile distilled water, dilute 1.5:500 in
PNM buffer.
- Biotinylated goat anti-avidin (Vector), 500 Ilg/ml in sterile distilled
water, dilute 1:100 in PNM buffer.
- DAPI stock solution (10 Ilg/ml): add 1 mg DAPI to 100 ml2xSSC and
store protected from the light at 4°C for at least 3 months.
- DAPI counterstain solution (0.1 Ilg/ml): add 50 rnl 2xSSC to SOO III
DAPI stock solution. Prepare fresh daily.
- Denaturation buffer: add 70 ml formamide to a solution of 10 ml
20xSSC and 20 ml distilled water and mix thoroughly. Adjust pH
to 7.5 with 2 N HCl and store at 4°C for 1 week.
- Deoxythymidintriphosphate (dTTP) (Sigma), 100 mM solution pH 7.0,
dilute with sterile distilled water to a final concentration of 1 mM.
- 70% ethanol: Mix 3S0 ml ethanol and 150 ml distilled water. Store at
room temperature.
- 8S% ethanol: Mix 425 ml ethanol and 75 ml distilled water. Store at
room temperature.
- 90% ethanol: Mix 450 ml ethanol and 50 rnl distilled water. Store at
room temperature.
- Fl detection reagent: For each slide, add 199 III 4xT solution to 1 III
rabbit anti-FITC antibody, mix gently and incubate at room temperature protected from the light for 10 min.
- F2 detection reagent: For each slide, add 198 III 4xT solution to FITClabeled goat anti-rabbit antibody, mix gently and incubate at room
temperature protected from the light for 10 min.
- FITC labeled goat anti-rabbit IgG antibodies (Vector)
- FITC-Iabeled goat anti-mouse (Vector), 1.5 mg/rnl in sterile distilled
water, dilute 1:300 in PNM buffer.
- Formaldehyde solution: add 2 ml formaldehyde (37%, Sigma) to 72 ml
1x PBS/MgClz. Prepare fresh daily.
- 50% formamide/O.S x SSC: add 50 ml formamide to a solution of 25 ml
2xSSC and 25 ml distilled water and mix thoroughly. Adjust pH to 7.5
with 2 N HCl and store at 4°C for 1 week.
HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
- Avidin-Cy5 (BDS), 1 mg/ml in sterile distilled water, dilute 1.5:500 in
PNM buffer.
- Biotinylated goat anti-avidin (Vector), 500 Ilg/ml in sterile distilled
water, dilute 1:100 in PNM buffer.
- DAPI stock solution (10 Ilg/ml): add 1 mg DAPI to 100 ml2xSSC and
store protected from the light at 4°C for at least 3 months.
- DAPI counterstain solution (0.1 Ilg/ml): add 50 rnl 2xSSC to SOO III
DAPI stock solution. Prepare fresh daily.
- Denaturation buffer: add 70 ml formamide to a solution of 10 ml
20xSSC and 20 ml distilled water and mix thoroughly. Adjust pH
to 7.5 with 2 N HCl and store at 4°C for 1 week.
- Deoxythymidintriphosphate (dTTP) (Sigma), 100 mM solution pH 7.0,
dilute with sterile distilled water to a final concentration of 1 mM.
- 70% ethanol: Mix 3S0 ml ethanol and 150 ml distilled water. Store at
room temperature.
- 8S% ethanol: Mix 425 ml ethanol and 75 ml distilled water. Store at
room temperature.
- 90% ethanol: Mix 450 ml ethanol and 50 rnl distilled water. Store at
room temperature.
- Fl detection reagent: For each slide, add 199 III 4xT solution to 1 III
rabbit anti-FITC antibody, mix gently and incubate at room temperature protected from the light for 10 min.
- F2 detection reagent: For each slide, add 198 III 4xT solution to FITClabeled goat anti-rabbit antibody, mix gently and incubate at room
temperature protected from the light for 10 min.
- FITC labeled goat anti-rabbit IgG antibodies (Vector)
- FITC-Iabeled goat anti-mouse (Vector), 1.5 mg/rnl in sterile distilled
water, dilute 1:300 in PNM buffer.
- Formaldehyde solution: add 2 ml formaldehyde (37%, Sigma) to 72 ml
1x PBS/MgClz. Prepare fresh daily.
- 50% formamide/O.S x SSC: add 50 ml formamide to a solution of 25 ml
2xSSC and 25 ml distilled water and mix thoroughly. Adjust pH to 7.5
with 2 N HCl and store at 4°C for 1 week.