removed from –80ºC and allowed to come to room temperature.
Slides are labeled and pretreated through a series of solutions,
including proteinaseK, triethanolamine, acetic anhydride and 2X
SSC, followed by dehydration through a graded series of ethanols.
33P-UTP riboprobes are generated by in vitro transcription using
approximately 0.5 µg of DNA template. Following column
purification probes are counted using a scintillation counter to
determine incorporation of 33P-UTP. A probe cocktail containing
50% formamide, 50% 2X hybridization buffer, 0.1% SDS and 1 x 10
6 cpm of probe are added to each slide. Slides are coverslipped
and incubated in a humid chamber overnight at 60ºC.
The following day, slides are rinsed through a series of post-hybridization
washes, which include, 2X SSC/formamide, 2XSSC, Rnase A, 2XSSC and
0.2X SSC, followed by dehydration through a series of graded ethanols.
Following air-drying, slides are taped into a film cassette
and exposed to Biomax MR film overnight.
The following day slides
are dipped in Kodak NTB2 emulsion. Once air-dried, slides
are boxed, wrapped in foil and stored at 4ºC. Based on the
results of the autoradiography, slides are exposed to the emulsion
for 1, 3 or 7 days. Slides are developed in Kodak D-19 Developer,
rinsed in water and fixed using Kodak Fixer. Following overnight
rinse in running water slides are dehydrated, coverslipped and allowed
to air-dry for one week.