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Immunocytochemistry, Electron Tomography, and Energy Dispersive X-ray Spectroscopy (EDXS) on Cryosections of Human Cancer Cells Doped with Stimuli Responsive Polymeric Nanogels Loaded with Iron Oxide Nanoparticles

  • Roberto Marotta
  • A. Falqui
  • A. Curcio
  • A. Quarta
  • Teresa Pellegrino
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1025)

Abstract

The cryosectioning technique is an alternative method for preparing biological material for Transmission Electron Microscopy (TEM). We have applied this technique to study the mechanism of cell internalization of stimuli-responsive polymeric nanogels exploited as cargo nanovectors. With respect to conventional TEM processing, cryosectioning technique better preserves the morphology of solvent-sensitive nanogels and enhances the visibility of membrane-bounded organelles inside the cell cytoplasm. In this chapter we describe the protocols we have established to perform Electron Microscopy (EM)-immunocytochemistry, Electron Tomography (ET), and Energy Dispersive X-ray Spectroscopy (EDXS) chemical analysis in Scanning TEM (STEM) on cryosections of HeLa cells treated with pH-responsive nanogels hosting short interference RNA (siRNAs) and iron oxide nanoparticles (IONPs).

Key words

Tokuyasu cryosectioning technique Immunocytochemistry Electron tomography (ET) Energy dispersive X-ray spectroscopy (EDXS) chemical analysis Scanning transmission electron microscopy (STEM) Stimuli-responsive polymeric nanoparticles Iron oxide nanoparticles (IONPs) loaded nanogel HeLa 

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Roberto Marotta
    • 1
  • A. Falqui
    • 1
  • A. Curcio
    • 1
  • A. Quarta
    • 1
    • 2
  • Teresa Pellegrino
    • 1
    • 2
  1. 1.Istituto Italiano di TecnologiaGenoaItaly
  2. 2.National Nanotechnology Laboratory of CNR-NANOLecceItaly

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