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Detecting Gastrin-Releasing Peptide Receptor by in situ PCR on Archived,,,Tissue

overnight at 37C. (We place the slides and a moist paper towel in a sealed plastic container in an incubator.) The next day, slides are washed by immersion in PBS for 5 minutes and dehydrated in graded ethanols as described.

Reverse transcription. RNA is reverse transcribed to cDNA in a 40l mixture containing 20 mM Tris-HCL (ph 8.4), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate (dNTP), 100 ng of selected reverse primer (we use 20 mers) and 2 U of reverse transcriptase at 42C for 45 minutes in a humidified chamber. Following reverse transcription, the slides are washed by immersion in PBS and dehydrated in graded ethanols as described.

PCR amplification. PCR amplification is performed in a 100l mixture containing 20 mM Trishcl (ph 8.3), 50 mM kcl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 mM dntps, 15% glycerol and 100 ng of each primer. Primers should be designed to yield a product less than 300 bp in size, and we use the same reverse primer as used for reverse transcription. This reaction mixture is applied to each tissue slice and covered using the resealable GeneFrame product. Amplification is then performed using the in situ adapter for the RoboCycler 40 Temperature Cycler. The following conditions are used: 96C for 2 minutes, 40 cycles at 96C for 30 seconds, 52C for 30 seconds, 74C for 60 seconds and one cycle at 74C for 5 minutes. After cycling, the slices are washed by immersion for 5 minutes in PBS and then dehydrated in graded ethanols as described.

Hybridization. The probe is readily generated by PCR using digoxigeninlabeled primers. In situ PCR amplification products are then detected by hybridization to digoxigeninlabeled DNA probes9 in hybridization buffer (2x SSC, 50% formamide, 7.5% dextran sulfate and 100 to 200 ng of probe). The hybridization solution is added to the slices moun
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