NETs detection and quantification in paraffin embedded samples using confocal microscopy

Detection and quantification of Neutrophil Extracellular Traps (NETs) in tissue samples has become a topic of great interest to understand their pathological role in various diseases. We describe a semi-automatic method of visualization and quantification of NETs in paraffin-embedded intracoronary thrombus aspirate samples. This study is based on colocalization of myeloperoxidase (MPO) and citrullinated histone 3 (H3Cit) as hallmark of the presence of NETs. For the analysis we used the confocal immunofluorescence microscopy technology to quantify the number of fields and the total area (in μm2) containing NETs in each thrombus sample. This observer-independent quantification method could be a useful tool to standardize the study of NETs in paraffin-embedded tissues, enabling comparison of results among different laboratories.