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 |
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| Institution: | 1. School of Medical Sciences (Discipline of Anatomy and Histology) – The Bosch Institute, The University of Sydney, NSW 2006, Australia;2. Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006, Australia;1. Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, MO;2. Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, St. Louis, MO;1. Department of Paediatrics, University of Calgary Faculty of Medicine and Alberta Children’s Hospital Research Institute, Calgary, Alberta, Canada;2. Department of Pathology (Neuropathology), University of Calgary Faculty of Medicine and Alberta Children’s Hospital Research Institute, Calgary, Alberta, Canada;3. Department of Pathology (Paediatric), University of Calgary Faculty of Medicine and Alberta Children’s Hospital Research Institute, Calgary, Alberta, Canada;4. Department of Clinical Neurosciences, University of Calgary Faculty of Medicine and Alberta Children’s Hospital Research Institute, Calgary, Alberta, Canada;1. Department of Medicinal Chemistry and Natural Medicine Chemistry, Department of Pharmacognosy, College of Pharmacy, Harbin Medical University, Harbin 150081, China;2. State-Province Key Laboratory of Biomedicine-Pharmaceutics of China, Harbin Medical University, Harbin 150081, China;3. Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China;4. Department of Ultrasound, The Second Affiliated Hospital of Harbin Medical University, Harbin 150081, China;1. Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany;2. Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany;3. Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary;4. School of Engineering and Science, Jacobs University Bremen, Bremen, Germany |
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| 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. |
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| Keywords: | Conventional electron microscopy Combined and correlative imaging Coated pits Clathrin-coated vesicles Electron tomography Epithelial transport Vesicles |
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