---
_id: '9756'
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.
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.).
alternative_title:
- Neuromethods
article_processing_charge: No
author:
- first_name: Walter
  full_name: Kaufmann, Walter
  id: 3F99E422-F248-11E8-B48F-1D18A9856A87
  last_name: Kaufmann
  orcid: 0000-0001-9735-5315
- first_name: David
  full_name: Kleindienst, David
  id: 42E121A4-F248-11E8-B48F-1D18A9856A87
  last_name: Kleindienst
- first_name: Harumi
  full_name: Harada, Harumi
  id: 2E55CDF2-F248-11E8-B48F-1D18A9856A87
  last_name: Harada
  orcid: 0000-0001-7429-7896
- first_name: Ryuichi
  full_name: Shigemoto, Ryuichi
  id: 499F3ABC-F248-11E8-B48F-1D18A9856A87
  last_name: Shigemoto
  orcid: 0000-0001-8761-9444
citation:
  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>'
  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>'
  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>.'
  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.'
  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>.
  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.
date_created: 2021-07-30T09:34:56Z
date_published: 2021-07-27T00:00:00Z
date_updated: 2024-03-25T23:30:16Z
day: '27'
ddc:
- '573'
department:
- _id: RySh
- _id: EM-Fac
doi: 10.1007/978-1-0716-1522-5_19
ec_funded: 1
has_accepted_license: '1'
intvolume: '       169'
keyword:
- 'Freeze-fracture replica: Deep learning'
- Immunogold labeling
- Integral membrane protein
- Electron microscopy
language:
- iso: eng
month: '07'
oa_version: None
page: 267-283
place: New York
project:
- _id: 25CA28EA-B435-11E9-9278-68D0E5697425
  call_identifier: H2020
  grant_number: '694539'
  name: 'In situ analysis of single channel subunit composition in neurons: physiological
    implication in synaptic plasticity and behaviour'
- _id: 25CBA828-B435-11E9-9278-68D0E5697425
  call_identifier: H2020
  grant_number: '720270'
  name: Human Brain Project Specific Grant Agreement 1 (HBP SGA 1)
publication: ' Receptor and Ion Channel Detection in the Brain'
publication_identifier:
  eisbn:
  - '9781071615225'
  isbn:
  - '9781071615218'
publication_status: published
publisher: Humana
quality_controlled: '1'
related_material:
  record:
  - id: '9562'
    relation: dissertation_contains
    status: public
series_title: Neuromethods
status: public
title: High-Resolution localization and quantitation of membrane proteins by SDS-digested
  freeze-fracture replica labeling (SDS-FRL)
type: book_chapter
user_id: D865714E-FA4E-11E9-B85B-F5C5E5697425
volume: 169
year: '2021'
...
---
_id: '8586'
abstract:
- lang: eng
  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.
acknowledged_ssus:
- _id: ScienComp
- _id: LifeSc
- _id: Bio
- _id: EM-Fac
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.
article_number: '107633'
article_processing_charge: Yes (via OA deal)
article_type: original
author:
- first_name: Florian
  full_name: Fäßler, Florian
  id: 404F5528-F248-11E8-B48F-1D18A9856A87
  last_name: Fäßler
  orcid: 0000-0001-7149-769X
- first_name: Bettina
  full_name: Zens, Bettina
  id: 45FD126C-F248-11E8-B48F-1D18A9856A87
  last_name: Zens
- first_name: Robert
  full_name: Hauschild, Robert
  id: 4E01D6B4-F248-11E8-B48F-1D18A9856A87
  last_name: Hauschild
  orcid: 0000-0001-9843-3522
- first_name: Florian KM
  full_name: Schur, Florian KM
  id: 48AD8942-F248-11E8-B48F-1D18A9856A87
  last_name: Schur
  orcid: 0000-0003-4790-8078
citation:
  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>
  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>
  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>.
  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.
  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>.
  short: F. Fäßler, B. Zens, R. Hauschild, F.K. Schur, Journal of Structural Biology
    212 (2020).
date_created: 2020-09-29T13:24:06Z
date_published: 2020-12-01T00:00:00Z
date_updated: 2024-03-25T23:30:04Z
day: '01'
ddc:
- '570'
department:
- _id: FlSc
doi: 10.1016/j.jsb.2020.107633
external_id:
  isi:
  - '000600997800008'
file:
- access_level: open_access
  checksum: c48cbf594e84fc2f91966ffaafc0918c
  content_type: application/pdf
  creator: dernst
  date_created: 2020-12-10T14:01:10Z
  date_updated: 2020-12-10T14:01:10Z
  file_id: '8937'
  file_name: 2020_JourStrucBiology_Faessler.pdf
  file_size: 7076870
  relation: main_file
  success: 1
file_date_updated: 2020-12-10T14:01:10Z
has_accepted_license: '1'
intvolume: '       212'
isi: 1
issue: '3'
keyword:
- electron microscopy
- cryo-EM
- EM sample preparation
- 3D printing
- cell culture
language:
- iso: eng
month: '12'
oa: 1
oa_version: Published Version
project:
- _id: 9B954C5C-BA93-11EA-9121-9846C619BF3A
  grant_number: P33367
  name: Structure and isoform diversity of the Arp2/3 complex
- _id: 059B463C-7A3F-11EA-A408-12923DDC885E
  name: NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria
publication: Journal of Structural Biology
publication_identifier:
  issn:
  - 1047-8477
publication_status: published
publisher: Elsevier
quality_controlled: '1'
related_material:
  record:
  - id: '14592'
    relation: used_in_publication
    status: public
  - id: '12491'
    relation: dissertation_contains
    status: public
scopus_import: '1'
status: public
title: 3D printed cell culture grid holders for improved cellular specimen preparation
  in cryo-electron microscopy
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 212
year: '2020'
...
---
_id: '11222'
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).
article_number: A3.27
article_processing_charge: No
author:
- first_name: Olena
  full_name: Kim, Olena
  id: 3F8ABDDA-F248-11E8-B48F-1D18A9856A87
  last_name: Kim
- first_name: Carolina
  full_name: Borges Merjane, Carolina
  id: 4305C450-F248-11E8-B48F-1D18A9856A87
  last_name: Borges Merjane
  orcid: 0000-0003-0005-401X
- first_name: Peter M
  full_name: Jonas, Peter M
  id: 353C1B58-F248-11E8-B48F-1D18A9856A87
  last_name: Jonas
  orcid: 0000-0001-5001-4804
citation:
  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>'
  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>.
  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.'
  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>.
  short: O. Kim, C. Borges Merjane, P.M. Jonas, in:, Intrinsic Activity, Austrian
    Pharmacological Society, 2019.
conference:
  end_date: 2019-09-27
  location: Innsbruck, Austria
  name: 'ANA: Austrian Neuroscience Association ; APHAR: Austrian Pharmacological
    Society'
  start_date: 2019-09-25
date_created: 2022-04-20T15:06:05Z
date_published: 2019-09-11T00:00:00Z
date_updated: 2024-03-25T23:30:04Z
day: '11'
department:
- _id: PeJo
doi: 10.25006/ia.7.s1-a3.27
ec_funded: 1
intvolume: '         7'
issue: Suppl. 1
keyword:
- hippocampus
- mossy fibers
- readily releasable pool
- electron microscopy
language:
- iso: eng
main_file_link:
- open_access: '1'
  url: https://www.intrinsicactivity.org/2019/7/S1/A3.27/
month: '09'
oa: 1
oa_version: Published Version
project:
- _id: 25B7EB9E-B435-11E9-9278-68D0E5697425
  call_identifier: H2020
  grant_number: '692692'
  name: Biophysics and circuit function of a giant cortical glumatergic synapse
- _id: 25BAF7B2-B435-11E9-9278-68D0E5697425
  call_identifier: H2020
  grant_number: '708497'
  name: Presynaptic calcium channels distribution and impact on coupling at the hippocampal
    mossy fiber synapse
- _id: 25C3DBB6-B435-11E9-9278-68D0E5697425
  call_identifier: FWF
  grant_number: W01205
  name: Zellkommunikation in Gesundheit und Krankheit
- _id: 25C5A090-B435-11E9-9278-68D0E5697425
  call_identifier: FWF
  grant_number: Z00312
  name: The Wittgenstein Prize
publication: Intrinsic Activity
publication_identifier:
  issn:
  - 2309-8503
publication_status: published
publisher: Austrian Pharmacological Society
quality_controlled: '1'
related_material:
  record:
  - id: '11196'
    relation: dissertation_contains
    status: public
status: public
title: Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals
  by electron microscopy
type: conference_abstract
user_id: 8b945eb4-e2f2-11eb-945a-df72226e66a9
volume: 7
year: '2019'
...