Summary Molecular diagnostics in personalized medicine increasingly relies on the combination of a variety of analytical technologies to characterize individual diseases and to select patients for targeted therapies. The gold standard for tissue-based diagnostics is fixation in formalin and embedding in paraffin, which results in excellent preservation of morphology but negatively impacts on a variety of molecular assays. The formalin-free, non-cross-linking PAXgene tissue system preserves morphology in a similar way to formalin, but also preserves biomolecules essentially in a similar way to cryopreservation, which markedly widens the spectrum, sensitivity and accuracy of molecular analytics. In this study, we have developed and tested a protocol for PAXgene-fixed and paraffin-embedded tissues for fluorescent in situ hybridization (FISH). The implementation of a 24-h formalin postfixation step of slides from PAXgene-fixed and paraffin-embedded tissues allowed us to use the assays approved for formalin-fixed and paraffin-embedded tissues. The equivalence of the methodologies was demonstrated by FISH analysis of HER2 amplification in breast cancer cases. The 24-h postfixation step of the slides used for FISH can be well integrated in the routine diagnostic workflow and allows the remaining PAXgene-fixed and paraffin-embedded tissue to be used for further molecular testing.