NETs detection and quantification in paraffin embedded samples using confocal microscopy

Abstract 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 μm 2 ) 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.