[{"article_processing_charge":"No","oa_version":"None","title":"High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL)","publication_status":"published","publication_identifier":{"isbn":["9781071615218"],"eisbn":["9781071615225"]},"acknowledgement":"This work was supported by the European Union (European Research Council Advanced grant no. 694539 and Human Brain Project Ref. 720270 to R. S.) and the Austrian Academy of Sciences (DOC fellowship to D.K.).","doi":"10.1007/978-1-0716-1522-5_19","_id":"9756","date_created":"2021-07-30T09:34:56Z","abstract":[{"lang":"eng","text":"High-resolution visualization and quantification of membrane proteins contribute to the understanding of their functions and the roles they play in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively the two-dimensional distribution of transmembrane proteins and their tightly associated proteins. During treatment with SDS, intracellular organelles and proteins not anchored to the replica are dissolved, whereas integral membrane proteins captured and stabilized by carbon/platinum deposition remain on the replica. Their intra- and extracellular domains become exposed on the surface of the replica, facilitating the accessibility of antibodies and, therefore, providing higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples, and optimization of the SDS treatment to increase the labeling efficiency for quantification of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing deep learning algorithms."}],"place":"New York","year":"2021","citation":{"mla":"Kaufmann, Walter, et al. “High-Resolution Localization and Quantitation of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).” <i> Receptor and Ion Channel Detection in the Brain</i>, vol. 169, Humana, 2021, pp. 267–83, doi:<a href=\"https://doi.org/10.1007/978-1-0716-1522-5_19\">10.1007/978-1-0716-1522-5_19</a>.","apa":"Kaufmann, W., Kleindienst, D., Harada, H., &#38; Shigemoto, R. (2021). High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In <i> Receptor and Ion Channel Detection in the Brain</i> (Vol. 169, pp. 267–283). New York: Humana. <a href=\"https://doi.org/10.1007/978-1-0716-1522-5_19\">https://doi.org/10.1007/978-1-0716-1522-5_19</a>","short":"W. Kaufmann, D. Kleindienst, H. Harada, R. Shigemoto, in:,  Receptor and Ion Channel Detection in the Brain, Humana, New York, 2021, pp. 267–283.","chicago":"Kaufmann, Walter, David Kleindienst, Harumi Harada, and Ryuichi Shigemoto. “High-Resolution Localization and Quantitation of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL).” In <i> Receptor and Ion Channel Detection in the Brain</i>, 169:267–83. Neuromethods. New York: Humana, 2021. <a href=\"https://doi.org/10.1007/978-1-0716-1522-5_19\">https://doi.org/10.1007/978-1-0716-1522-5_19</a>.","ama":"Kaufmann W, Kleindienst D, Harada H, Shigemoto R. High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In: <i> Receptor and Ion Channel Detection in the Brain</i>. Vol 169. Neuromethods. New York: Humana; 2021:267-283. doi:<a href=\"https://doi.org/10.1007/978-1-0716-1522-5_19\">10.1007/978-1-0716-1522-5_19</a>","ieee":"W. Kaufmann, D. Kleindienst, H. Harada, and R. Shigemoto, “High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL),” in <i> Receptor and Ion Channel Detection in the Brain</i>, vol. 169, New York: Humana, 2021, pp. 267–283.","ista":"Kaufmann W, Kleindienst D, Harada H, Shigemoto R. 2021.High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL). In:  Receptor and Ion Channel Detection in the Brain. Neuromethods, vol. 169, 267–283."},"related_material":{"record":[{"id":"9562","relation":"dissertation_contains","status":"public"}]},"volume":169,"series_title":"Neuromethods","user_id":"D865714E-FA4E-11E9-B85B-F5C5E5697425","author":[{"last_name":"Kaufmann","first_name":"Walter","full_name":"Kaufmann, Walter","orcid":"0000-0001-9735-5315","id":"3F99E422-F248-11E8-B48F-1D18A9856A87"},{"id":"42E121A4-F248-11E8-B48F-1D18A9856A87","full_name":"Kleindienst, David","first_name":"David","last_name":"Kleindienst"},{"full_name":"Harada, Harumi","id":"2E55CDF2-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7429-7896","last_name":"Harada","first_name":"Harumi"},{"last_name":"Shigemoto","first_name":"Ryuichi","full_name":"Shigemoto, Ryuichi","orcid":"0000-0001-8761-9444","id":"499F3ABC-F248-11E8-B48F-1D18A9856A87"}],"publication":" Receptor and Ion Channel Detection in the Brain","ec_funded":1,"date_updated":"2024-03-25T23:30:16Z","department":[{"_id":"RySh"},{"_id":"EM-Fac"}],"intvolume":"       169","quality_controlled":"1","day":"27","page":"267-283","project":[{"name":"In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behaviour","_id":"25CA28EA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","grant_number":"694539"},{"call_identifier":"H2020","grant_number":"720270","_id":"25CBA828-B435-11E9-9278-68D0E5697425","name":"Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)"}],"has_accepted_license":"1","date_published":"2021-07-27T00:00:00Z","type":"book_chapter","publisher":"Humana","status":"public","month":"07","keyword":["Freeze-fracture replica: Deep learning","Immunogold labeling","Integral membrane protein","Electron microscopy"],"alternative_title":["Neuromethods"],"language":[{"iso":"eng"}],"ddc":["573"]},{"_id":"8586","file":[{"file_size":7076870,"date_updated":"2020-12-10T14:01:10Z","date_created":"2020-12-10T14:01:10Z","relation":"main_file","access_level":"open_access","success":1,"file_name":"2020_JourStrucBiology_Faessler.pdf","checksum":"c48cbf594e84fc2f91966ffaafc0918c","creator":"dernst","file_id":"8937","content_type":"application/pdf"}],"date_created":"2020-09-29T13:24:06Z","article_number":"107633","year":"2020","citation":{"ieee":"F. Fäßler, B. Zens, R. Hauschild, and F. K. Schur, “3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy,” <i>Journal of Structural Biology</i>, vol. 212, no. 3. Elsevier, 2020.","ista":"Fäßler F, Zens B, Hauschild R, Schur FK. 2020. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. Journal of Structural Biology. 212(3), 107633.","ama":"Fäßler F, Zens B, Hauschild R, Schur FK. 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. <i>Journal of Structural Biology</i>. 2020;212(3). doi:<a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">10.1016/j.jsb.2020.107633</a>","mla":"Fäßler, Florian, et al. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” <i>Journal of Structural Biology</i>, vol. 212, no. 3, 107633, Elsevier, 2020, doi:<a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">10.1016/j.jsb.2020.107633</a>.","chicago":"Fäßler, Florian, Bettina Zens, Robert Hauschild, and Florian KM Schur. “3D Printed Cell Culture Grid Holders for Improved Cellular Specimen Preparation in Cryo-Electron Microscopy.” <i>Journal of Structural Biology</i>. Elsevier, 2020. <a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">https://doi.org/10.1016/j.jsb.2020.107633</a>.","short":"F. Fäßler, B. Zens, R. Hauschild, F.K. Schur, Journal of Structural Biology 212 (2020).","apa":"Fäßler, F., Zens, B., Hauschild, R., &#38; Schur, F. K. (2020). 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy. <i>Journal of Structural Biology</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.jsb.2020.107633\">https://doi.org/10.1016/j.jsb.2020.107633</a>"},"abstract":[{"text":"Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.","lang":"eng"}],"title":"3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy","publication_status":"published","file_date_updated":"2020-12-10T14:01:10Z","oa_version":"Published Version","article_processing_charge":"Yes (via OA deal)","doi":"10.1016/j.jsb.2020.107633","scopus_import":"1","article_type":"original","publication_identifier":{"issn":["1047-8477"]},"acknowledgement":"This work was supported by the Austrian Science Fund (FWF, P33367) to FKMS. BZ acknowledges support by the Niederösterreich Fond. This research was also supported by the Scientific Service Units (SSU) of IST Austria through resources provided by Scientific Computing (SciComp), the Life Science Facility (LSF), the BioImaging Facility (BIF) and the Electron Microscopy Facility (EMF). We thank Georgi Dimchev (IST Austria) and Sonja Jacob (Vienna Biocenter Core Facilities) for testing our grid holders in different experimental setups and Daniel Gütl and the Kondrashov group (IST Austria) for granting us repeated access to their 3D printers. We also thank Jonna Alanko and the Sixt lab (IST Austria) for providing us HeLa cells, primary BL6 mouse tail fibroblasts, NIH 3T3 fibroblasts and human telomerase immortalised foreskin fibroblasts for our experiments. We are thankful to Ori Avinoam and William Wan for helpful comments on the manuscript and also thank Dorotea Fracchiolla (Art&Science) for illustrating the graphical abstract.","department":[{"_id":"FlSc"}],"publication":"Journal of Structural Biology","date_updated":"2024-03-25T23:30:04Z","intvolume":"       212","acknowledged_ssus":[{"_id":"ScienComp"},{"_id":"LifeSc"},{"_id":"Bio"},{"_id":"EM-Fac"}],"related_material":{"record":[{"id":"14592","relation":"used_in_publication","status":"public"},{"relation":"dissertation_contains","status":"public","id":"12491"}]},"oa":1,"volume":212,"author":[{"last_name":"Fäßler","first_name":"Florian","full_name":"Fäßler, Florian","orcid":"0000-0001-7149-769X","id":"404F5528-F248-11E8-B48F-1D18A9856A87"},{"id":"45FD126C-F248-11E8-B48F-1D18A9856A87","full_name":"Zens, Bettina","first_name":"Bettina","last_name":"Zens"},{"last_name":"Hauschild","first_name":"Robert","full_name":"Hauschild, Robert","orcid":"0000-0001-9843-3522","id":"4E01D6B4-F248-11E8-B48F-1D18A9856A87"},{"id":"48AD8942-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-4790-8078","full_name":"Schur, Florian KM","first_name":"Florian KM","last_name":"Schur"}],"user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","issue":"3","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","short":"CC BY (4.0)"},"project":[{"_id":"9B954C5C-BA93-11EA-9121-9846C619BF3A","name":"Structure and isoform diversity of the Arp2/3 complex","grant_number":"P33367"},{"name":"NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria","_id":"059B463C-7A3F-11EA-A408-12923DDC885E"}],"has_accepted_license":"1","quality_controlled":"1","day":"01","month":"12","isi":1,"status":"public","language":[{"iso":"eng"}],"ddc":["570"],"keyword":["electron microscopy","cryo-EM","EM sample preparation","3D printing","cell culture"],"date_published":"2020-12-01T00:00:00Z","external_id":{"isi":["000600997800008"]},"type":"journal_article","publisher":"Elsevier"},{"language":[{"iso":"eng"}],"keyword":["hippocampus","mossy fibers","readily releasable pool","electron microscopy"],"month":"09","status":"public","publisher":"Austrian Pharmacological Society","type":"conference_abstract","date_published":"2019-09-11T00:00:00Z","issue":"Suppl. 1","project":[{"grant_number":"692692","call_identifier":"H2020","_id":"25B7EB9E-B435-11E9-9278-68D0E5697425","name":"Biophysics and circuit function of a giant cortical glumatergic synapse"},{"call_identifier":"H2020","grant_number":"708497","_id":"25BAF7B2-B435-11E9-9278-68D0E5697425","name":"Presynaptic calcium channels distribution and impact on coupling at the hippocampal mossy fiber synapse"},{"_id":"25C3DBB6-B435-11E9-9278-68D0E5697425","name":"Zellkommunikation in Gesundheit und Krankheit","grant_number":"W01205","call_identifier":"FWF"},{"_id":"25C5A090-B435-11E9-9278-68D0E5697425","name":"The Wittgenstein Prize","grant_number":"Z00312","call_identifier":"FWF"}],"day":"11","quality_controlled":"1","intvolume":"         7","department":[{"_id":"PeJo"}],"date_updated":"2024-03-25T23:30:04Z","ec_funded":1,"publication":"Intrinsic Activity","author":[{"last_name":"Kim","first_name":"Olena","full_name":"Kim, Olena","id":"3F8ABDDA-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Borges Merjane","first_name":"Carolina","full_name":"Borges Merjane, Carolina","id":"4305C450-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-0005-401X"},{"last_name":"Jonas","first_name":"Peter M","full_name":"Jonas, Peter M","id":"353C1B58-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-5001-4804"}],"user_id":"8b945eb4-e2f2-11eb-945a-df72226e66a9","volume":7,"related_material":{"record":[{"relation":"dissertation_contains","status":"public","id":"11196"}]},"oa":1,"citation":{"short":"O. Kim, C. Borges Merjane, P.M. Jonas, in:, Intrinsic Activity, Austrian Pharmacological Society, 2019.","apa":"Kim, O., Borges Merjane, C., &#38; Jonas, P. M. (2019). Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy. In <i>Intrinsic Activity</i> (Vol. 7). Innsbruck, Austria: Austrian Pharmacological Society. <a href=\"https://doi.org/10.25006/ia.7.s1-a3.27\">https://doi.org/10.25006/ia.7.s1-a3.27</a>","chicago":"Kim, Olena, Carolina Borges Merjane, and Peter M Jonas. “Functional Analysis of the Docked Vesicle Pool in Hippocampal Mossy Fiber Terminals by Electron Microscopy.” In <i>Intrinsic Activity</i>, Vol. 7. Austrian Pharmacological Society, 2019. <a href=\"https://doi.org/10.25006/ia.7.s1-a3.27\">https://doi.org/10.25006/ia.7.s1-a3.27</a>.","mla":"Kim, Olena, et al. “Functional Analysis of the Docked Vesicle Pool in Hippocampal Mossy Fiber Terminals by Electron Microscopy.” <i>Intrinsic Activity</i>, vol. 7, no. Suppl. 1, A3.27, Austrian Pharmacological Society, 2019, doi:<a href=\"https://doi.org/10.25006/ia.7.s1-a3.27\">10.25006/ia.7.s1-a3.27</a>.","ama":"Kim O, Borges Merjane C, Jonas PM. Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy. In: <i>Intrinsic Activity</i>. Vol 7. Austrian Pharmacological Society; 2019. doi:<a href=\"https://doi.org/10.25006/ia.7.s1-a3.27\">10.25006/ia.7.s1-a3.27</a>","ieee":"O. Kim, C. Borges Merjane, and P. M. Jonas, “Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy,” in <i>Intrinsic Activity</i>, Innsbruck, Austria, 2019, vol. 7, no. Suppl. 1.","ista":"Kim O, Borges Merjane C, Jonas PM. 2019. Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy. Intrinsic Activity. ANA: Austrian Neuroscience Association ; APHAR: Austrian Pharmacological Society vol. 7, A3.27."},"article_number":"A3.27","year":"2019","conference":{"location":"Innsbruck, Austria","name":"ANA: Austrian Neuroscience Association ; APHAR: Austrian Pharmacological Society","end_date":"2019-09-27","start_date":"2019-09-25"},"date_created":"2022-04-20T15:06:05Z","_id":"11222","main_file_link":[{"open_access":"1","url":"https://www.intrinsicactivity.org/2019/7/S1/A3.27/"}],"doi":"10.25006/ia.7.s1-a3.27","publication_identifier":{"issn":["2309-8503"]},"acknowledgement":"This work was supported by the ERC and EU Horizon 2020 (ERC 692692; MSC-IF 708497) and FWF Z 312-B27 Wittgenstein award; W 1205-B09).","title":"Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy","publication_status":"published","article_processing_charge":"No","oa_version":"Published Version"}]