7 In Situ Hybridization for RNA: Radioactive DNA Probe
101
Materials
Laboratory for radioisotope, photo darkroom, centrifugal se- Instruments
parator( for Eppendorf tube), speed vacuum concentrator (Sar- and machines
vant), water bath (30x30xSOcm), small water bath (2Scm diameter), aluminum cake pan (24x24xScm) (G&S Metal Products
Co., Inc., Cleveland, OH), Nensorb 20 column (Dupon), Eppendorf tube, column stand, expanded polystyrene box (S liter volume), liquid scintillation counter, slide glass (highquality), distilled water, sliding microtome, artificial incubator, hot plate,
sterilizer, Caplin jar, dark specimen box (for 2S slides),
22x26mm cover glass (Corning Glass Works, Corning, NY).
Specific cDNA probe (300bp-lkbp fragment, 20-S0 A), 3H_ la- Chemical
beled dTTP or 35S_ labeled dTTP (Dupon), Sx nick translation reagents
buffer (SOmM Tris-HCI, pH 7.S, SmM MgClz, lOmM B-mercapto ethanol, SO )lg/ml BSA), unlabeled dNTP (O.3mM each of
dATP, dCTP, dGTP in SOmM Tris-HCI, pH 7.6), 0.03 )lg/ml
DNase I (Boehringer Mannheim Biochemicals, Indianapolis,
IN), Escherichia coli DNA polymerase I (Boehringer Mannheim
Biochemicals, Indianapolis, IN), 0.2M EDT A, salmon sperm
DNA (80)lg/ml) (Funakoshi), pronase E (Type XXV; Calbiochern), formamide, paraffin for embedding, 3-aminopropyltriethoxysilane (Aldolich), 4% paraformaldehyde (EM grade,
TAAB) / phosphate buffered saline (PBS) fixation buffer, ethanol (for HPLC), xylene (for HPLC), rubber cement (Carter's Ink
Co., Waltham, MA), siliconization solution (Sigma coat; Sigma),
petroleum ether, Kodak NTB-2 nuclear track emulsion, Harris'
alum hematoxylin (Harleco, Gibbstown, NJ), permount (Fisher
Science), phosphate buffered saline (PBS), TNEB-DS mixture
(20mM Tris-HCI, 0.3 M N aCI, 2mM EDT A, pH 7.0, 2x Denhardt's
mixture, 20% dextran sulfate), 2M dithiothreitol (DTT) solution,
methanol (for HPLC), yeast t-RNA (SOO )lg/ml), 0.6 M ammonium acetate, pH 7.0, Kodak D-19 developer, dextran sulfate
(Pharmacia P-L Biochemicals, Piscataway, NJ), Denhardt's mixture, 20x Standard sodium citrate buffer (SSC).
The requirements of ISH have limitations as to the radioisotope Radioisotope
that can be used. Although it is said that the ideal radioisotope labels
would allow a rapid result with excellent localization and the
minimum of safety problem, it would be a pure B-emitter
101
Materials
Laboratory for radioisotope, photo darkroom, centrifugal se- Instruments
parator( for Eppendorf tube), speed vacuum concentrator (Sar- and machines
vant), water bath (30x30xSOcm), small water bath (2Scm diameter), aluminum cake pan (24x24xScm) (G&S Metal Products
Co., Inc., Cleveland, OH), Nensorb 20 column (Dupon), Eppendorf tube, column stand, expanded polystyrene box (S liter volume), liquid scintillation counter, slide glass (highquality), distilled water, sliding microtome, artificial incubator, hot plate,
sterilizer, Caplin jar, dark specimen box (for 2S slides),
22x26mm cover glass (Corning Glass Works, Corning, NY).
Specific cDNA probe (300bp-lkbp fragment, 20-S0 A), 3H_ la- Chemical
beled dTTP or 35S_ labeled dTTP (Dupon), Sx nick translation reagents
buffer (SOmM Tris-HCI, pH 7.S, SmM MgClz, lOmM B-mercapto ethanol, SO )lg/ml BSA), unlabeled dNTP (O.3mM each of
dATP, dCTP, dGTP in SOmM Tris-HCI, pH 7.6), 0.03 )lg/ml
DNase I (Boehringer Mannheim Biochemicals, Indianapolis,
IN), Escherichia coli DNA polymerase I (Boehringer Mannheim
Biochemicals, Indianapolis, IN), 0.2M EDT A, salmon sperm
DNA (80)lg/ml) (Funakoshi), pronase E (Type XXV; Calbiochern), formamide, paraffin for embedding, 3-aminopropyltriethoxysilane (Aldolich), 4% paraformaldehyde (EM grade,
TAAB) / phosphate buffered saline (PBS) fixation buffer, ethanol (for HPLC), xylene (for HPLC), rubber cement (Carter's Ink
Co., Waltham, MA), siliconization solution (Sigma coat; Sigma),
petroleum ether, Kodak NTB-2 nuclear track emulsion, Harris'
alum hematoxylin (Harleco, Gibbstown, NJ), permount (Fisher
Science), phosphate buffered saline (PBS), TNEB-DS mixture
(20mM Tris-HCI, 0.3 M N aCI, 2mM EDT A, pH 7.0, 2x Denhardt's
mixture, 20% dextran sulfate), 2M dithiothreitol (DTT) solution,
methanol (for HPLC), yeast t-RNA (SOO )lg/ml), 0.6 M ammonium acetate, pH 7.0, Kodak D-19 developer, dextran sulfate
(Pharmacia P-L Biochemicals, Piscataway, NJ), Denhardt's mixture, 20x Standard sodium citrate buffer (SSC).
The requirements of ISH have limitations as to the radioisotope Radioisotope
that can be used. Although it is said that the ideal radioisotope labels
would allow a rapid result with excellent localization and the
minimum of safety problem, it would be a pure B-emitter