Successful alignment of H& E and AR. A, tissue section of a prostate biopsy, stained for H&E (20x magnification); B, the same tissue section stained for AR using TRF (40x). C, Successful alignment shown as an overlay of image B onto image A. The staining procedure was the following: first the tissue section was stained for AR and pictures acquired through TRF, then the AR was washed off, the section was stained for H&E and a new picture was acquired through brightfield microscopy. Considering that AR is the protein to be quantified, it is important that AR expression is preserved and therefore that the tissue is minimally stressed. Since the tissue is processed twice and this might alter its structure and protein content, we have performed AR as the first staining. H&E on the other hand did not seem to be highly influenced by intermediate steps.