256
WILLIAM M. STRAUSS
Materials
- PNA (PerSeptive Biosystems, Framingham, MA)
- 0.1% aqueous trifluoroacetic acid (TFA, Sigma, S1. Louis, MO)
- lOX phosphate-buffered saline (PBS) pH 7.2 (Life Technologies,
Gaithersburg, MD)
- 70%, 80%, 90%, and 100% ethanol series
- 37% formaldehyde (Fisher Scientific)
- 100% formamide (Fisher Scientific) for post-hybridization washing
- 100% deionized formamide (Oncor, Gaithersburg, MD)
- Tween-20 (Sigma, St. Louis, MO)
- 100 mM Tris-pH 7.2; 5 M NaCI
- 10% blocking reagent (Boehringer-Mannheim, Indianapolis, IN)
- f..lglml of 4,6-diamidino-2-phenylindole-dehydrochloride (DAPI) (Oncor, Gaitherburg, MD)
- 0.6 f..lglml of propidium iodide (PI) (Oncor, Gaitherburg, MD)
Buffers - 1x hybridization mix: Add 35 ml of deionized formamide, 5 ml of 10%
blocking reagent, and 5 ml of 100 mM Tris pH 7.2. Adjust pH to 7.2 by
adding 1:5 diluted HCI. Aliquot into 0.5 ml each and store at minus
20°C for up to 6 months.
- 70% formamide 11 xPBS: For 5 slides, mix 105 ml offormamide, 15 ml
of lOx PBS, and 20 ml of dd. H 2 0. Bring pH to 7.2 by adding 1:10 diluted HCI and volume to 150 mi. Make freshly.
- TNT washing buffer: 50 mM Tris pH 7.51 150 mM NaClIO.05%(v/v)
Tween-20. Make freshly.
Equipment - Moist chamber
- Glass slides
- Glass coverslips (22x22, 24x30, 24x40 mm)
- 4 glass staining dishes
- Blunted forceps
WILLIAM M. STRAUSS
Materials
- PNA (PerSeptive Biosystems, Framingham, MA)
- 0.1% aqueous trifluoroacetic acid (TFA, Sigma, S1. Louis, MO)
- lOX phosphate-buffered saline (PBS) pH 7.2 (Life Technologies,
Gaithersburg, MD)
- 70%, 80%, 90%, and 100% ethanol series
- 37% formaldehyde (Fisher Scientific)
- 100% formamide (Fisher Scientific) for post-hybridization washing
- 100% deionized formamide (Oncor, Gaithersburg, MD)
- Tween-20 (Sigma, St. Louis, MO)
- 100 mM Tris-pH 7.2; 5 M NaCI
- 10% blocking reagent (Boehringer-Mannheim, Indianapolis, IN)
- f..lglml of 4,6-diamidino-2-phenylindole-dehydrochloride (DAPI) (Oncor, Gaitherburg, MD)
- 0.6 f..lglml of propidium iodide (PI) (Oncor, Gaitherburg, MD)
Buffers - 1x hybridization mix: Add 35 ml of deionized formamide, 5 ml of 10%
blocking reagent, and 5 ml of 100 mM Tris pH 7.2. Adjust pH to 7.2 by
adding 1:5 diluted HCI. Aliquot into 0.5 ml each and store at minus
20°C for up to 6 months.
- 70% formamide 11 xPBS: For 5 slides, mix 105 ml offormamide, 15 ml
of lOx PBS, and 20 ml of dd. H 2 0. Bring pH to 7.2 by adding 1:10 diluted HCI and volume to 150 mi. Make freshly.
- TNT washing buffer: 50 mM Tris pH 7.51 150 mM NaClIO.05%(v/v)
Tween-20. Make freshly.
Equipment - Moist chamber
- Glass slides
- Glass coverslips (22x22, 24x30, 24x40 mm)
- 4 glass staining dishes
- Blunted forceps