Figure 2.

Flow chart describing the application of in situ RT-PCR to tissue microarrays. Step 1: Slide preparation. Sections were deparaffinated by xylene washes, and celluar material permeabilised by limited proteinase K digestion; Step 2: amplification of reverse-transcribed cDNA using intron-spanning PCR primers (horizontal arrows) added in a single mix of reverse transcriptase, rTth polymerase and deoxyribonucleotides spiked with digoxygenin-labelled dUTP (black ovals); Step 3: visualisation of PCR products by binding to digoxygenin-specific gold-labelled antibodies (yellow dots), followed by silver nucleation around the bound gold particles (grey crescents).