In Situ Hybridization

Slides are 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.

For detailed in situ hybridization protocols please click on Links below:

•  Generation of Riboprobe

•  Hybridization

•  Post-Hybridization

•  Emulsion

•  Developer and Fixer