title: Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: a multidimensional view by means of correlative light and 3-D transmission electron microscopy.
abstract: Herein, we present a comparative analysis of a variety of chemical and physical fixation protocols for the specific visualisation of the membrane-bound vesicles (MBVs) in the Caco-2 colorectal cancer (CRC) cell line. In so doing, we validated the applicability of specific specimen preparation protocols for the preservation and contrasting of membrane-associated vesicles. Next, by employing the best respective chemical (GOT) and physical (SHPF) fixation methods for the application of transmission electron tomography and modelling we were able to characterise MBVs in three-dimensions and at the nanometer scale. In the second part of this study, we employ a correlative light and electron microscopy (CLEM) approach in order to determine which vesicular compartments are implicated in the uptake of FITC-BSA as a model protein drug. In so doing, we provide a solid foundation for future studies investigating chemotherapeutic drug uptake, transport and fate in cancer cell lines.
authors.affiliation: School of Medical Sciences (Discipline of Anatomy and Histology) - The Bosch Institute, The University of Sydney, NSW 2006, Australia. Electronic address: gerryshami@gmail.com.
authors.forename: Gerald
authors.initials: G
authors.lastname: Shami
authors.affiliation: School of Medical Sciences (Discipline of Anatomy and Histology) - The Bosch Institute, The University of Sydney, NSW 2006, Australia.
authors.forename: Delfine
authors.initials: D
authors.lastname: Cheng
authors.affiliation: School of Medical Sciences (Discipline of Anatomy and Histology) - The Bosch Institute, The University of Sydney, NSW 2006, Australia.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Henriquez
authors.affiliation: School of Medical Sciences (Discipline of Anatomy and Histology) - The Bosch Institute, The University of Sydney, NSW 2006, Australia; Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006, Australia. Electronic address: filip.braet@sydney.edu.au.
authors.forename: Filip
authors.initials: F
authors.lastname: Braet
mesh_terms: D000418:Albumins / Q000378:metabolism / Q000648:ultrastructure; D018938:Caco-2 Cells / Q000648:ultrastructure*; D002966:Clathrin / Q000378:metabolism / Q000648:ultrastructure; D018699:Coated Vesicles / Q000648:ultrastructure; D015925:Cryopreservation / Q000379:methods; D022162:Cytoplasmic Vesicles / Q000648:ultrastructure*; D005404:Fixatives; D005976:Glutaral; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D008853:Microscopy / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D009993:Osmium Tetroxide; D013634:Tannins; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D000418:Albumins; D002966:Clathrin; D005404:Fixatives; D013634:Tannins; D009993:Osmium Tetroxide; D005976:Glutaral
keywords: Clathrin-coated vesicles; Coated pits; Combined and correlative imaging; Conventional electron microscopy; Electron tomography; Epithelial transport; Vesicles