ted on the slides, and the slides are sealed and heated to 95C for 15 minutes and maintained at 37C overnight for hybridization. The next day, the hybridized slides are washed in 2.5% BSA in 0.2x SSC for 15 minutes at 42C, followed by PBS for 5 minutes. After washing, 100 l of a 1:200 dilution of alkaline phosphataseconjugated, antidigoxigenin antibody (BoehringerMannheim, Indianapolis, IN) are added to the slides, and the slides are incubated for 1 hour in a humidified chamber. The slides are then washed in PBS for 5 minutes and developed with NBT/BCIP solution. The reaction is stopped by washing in PBS, followed by dehydration of the tissue in graded ethanols (figure 2). The specimens are covered using Situ/Mount product. We avoid standard mounts because they can solubilize the stain, causing it to disappear over time.

figure 2

Controls. All specimens should be processed in triplicate, allowing for both positive and negative controls. The negative control is processed as described except that reverse transcriptase is excluded from the reaction mixture; the positive control slide is not treated with DNase I. The RoboCycler 40 Temperature Cycler in situ adapter system conveniently holds three standard microscope slides so that the experimental tissue can be processed simultaneously with positive and negative controls (data not shown).

Caveats. Subjective interpretation of immunohistochemistry to determine the number of positive cells should be avoided. Rather, an objective approach, such as quantitative PCR,¹³ should be used. Quantitative PCR can be performed on RNA extracted from a 20-M sample of freshly sectioned archival tissue.¹⁴ Finally, if
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