title: 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.
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).
authors.affiliation: Istituto Italiano di Tecnologia, Genoa, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Marotta
authors.forename: A
authors.initials: A
authors.lastname: Falqui
authors.forename: A
authors.initials: A
authors.lastname: Curcio
authors.forename: A
authors.initials: A
authors.lastname: Quarta
authors.forename: Teresa
authors.initials: T
authors.lastname: Pellegrino
mesh_terms: D016616:Cryoultramicrotomy / Q000379:methods*; D055032:Electron Microscope Tomography; D005290:Ferric Compounds / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D046529:Microscopy, Electron, Transmission; D008967:Molecular Biology / Q000379:methods; D000080385:Nanogels; D053758:Nanoparticles / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism*; D011094:Polyethyleneimine / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology*; D013052:Spectrometry, X-Ray Emission
chemical_list: D005290:Ferric Compounds; D000080385:Nanogels; D011108:Polymers; C413692:polyethylene glycol polyethyleneimine nanogel; C000499:ferric oxide; D011092:Polyethylene Glycols; D011094:Polyethyleneimine