NETs detection and quantification in paraffin embedded samples using confocal microscopy

A Santos; P Martín; A Blasco; J Solano; B Cózar; D García; J Goicolea; C Bellas; M J Coronado

doi:10.1016/j.micron.2018.07.002

NETs detection and quantification in paraffin embedded samples using confocal microscopy

Micron. 2018 Nov:114:1-7. doi: 10.1016/j.micron.2018.07.002. Epub 2018 Jul 18.

Authors

A Santos¹, P Martín², A Blasco³, J Solano⁴, B Cózar⁵, D García⁶, J Goicolea⁷, C Bellas⁸, M J Coronado⁹

Affiliations

¹ Molecular Pathology Laboratory, Instituto de Investigación Puerta de Hierro Segovia Arana (IDIPHISA), C/ Joaquín Rodrigo, 2, 28222 Majadahonda, Madrid, Spain. Electronic address: [email protected].
² Molecular Pathology Laboratory, Instituto de Investigación Puerta de Hierro Segovia Arana (IDIPHISA), C/ Joaquín Rodrigo, 2, 28222 Majadahonda, Madrid, Spain; Pathology Department, Hospital Universitario Puerta de Hierro, C/ Manuel de Falla, 1, 28222 Majadahonda, Madrid, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain. Electronic address: [email protected].
³ Cardiology Department, Hospital Universitario Puerta de Hierro, C/ Manuel de Falla, 1, 28222 Majadahonda, Madrid, Spain. Electronic address: [email protected].
⁴ Cardiology Department, Hospital Universitario Puerta de Hierro, C/ Manuel de Falla, 1, 28222 Majadahonda, Madrid, Spain. Electronic address: [email protected].
⁵ Cardiology Department, Hospital Universitario Puerta de Hierro, C/ Manuel de Falla, 1, 28222 Majadahonda, Madrid, Spain; Confocal Microscopy Unit, Instituto de Investigación Puerta de Hierro Segovia Arana (IDIPHISA), C/ Joaquín Rodrigo, 2, 28222 Majadahonda, Madrid, Spain. Electronic address: [email protected].
⁶ Pathology Department, Hospital Universitario Puerta de Hierro, C/ Manuel de Falla, 1, 28222 Majadahonda, Madrid, Spain. Electronic address: [email protected].
⁷ Cardiology Department, Hospital Universitario Puerta de Hierro, C/ Manuel de Falla, 1, 28222 Majadahonda, Madrid, Spain. Electronic address: [email protected].
⁸ Molecular Pathology Laboratory, Instituto de Investigación Puerta de Hierro Segovia Arana (IDIPHISA), C/ Joaquín Rodrigo, 2, 28222 Majadahonda, Madrid, Spain; Pathology Department, Hospital Universitario Puerta de Hierro, C/ Manuel de Falla, 1, 28222 Majadahonda, Madrid, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain. Electronic address: [email protected].
⁹ Confocal Microscopy Unit, Instituto de Investigación Puerta de Hierro Segovia Arana (IDIPHISA), C/ Joaquín Rodrigo, 2, 28222 Majadahonda, Madrid, Spain. Electronic address: [email protected].

PMID: 30028972
DOI: 10.1016/j.micron.2018.07.002

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 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.

Keywords: Image analysis; Inmunofluorescence; Microscopy; Neutrophil Extracellular Traps.

Publication types

Research Support, Non-U.S. Gov't