Objective: MicroRNAs are small noncoding RNAs that constitute a novel group of biomarkers with exciting functions in cell differentiation, proliferation and apoptosis by mediating degradation or destabilization of target mRNAs. miR-21 is highly prevalent in malignancies. Previous reports on miR-21 in situ hybridization (ISH) in colon and breast cancer identified expression in the stromal cell population that was related to recurrence and cell proliferation (Nielsen et al., Clin Exp Metastasis 2011, 28:27; Rask et al., APMIS 2011, 119:663) . Here, we present a histological assay platform that allows parallel localization of micro-RNAs and protein markers. Method: Routinely processed paraffin embedded breast cancer samples were analyzed. MicroRNA ISH probes were detected with fluorescent tyramine reagents and primary antibodies with compatible immunofluorescence. The reagents were introduced on a semi-automated platform, HistoFlex, which has a precise temperature control unit and utilizes continuous liquid flow. Results: By combination of miR-21 ISH and PDCD4 immunofluorescence we found differential expression in many but not all miR-21 positive cells. Using the HistoFlex, the double fluorescence assay was reduced from 7 to 2 h without loss of sensitivity and specificity. Conclusion: A 3-fold faster assay platform was developed and our in vivo findings support the assumption that PDCD4 is a target of miR-21.