Freeze‐Fracture Immunocytochemistry: Fracture‐Label and Label‐Fracture for the Localization of Membrane Proteins

Frederick W. K. Kan1

1 Department of Biomedical and Molecular Sciences, Faculty of Health Sciences, Queen's University, Kingston, Ontario
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.28
DOI:  10.1002/0471143030.cb0428s65
Online Posting Date:  December, 2014
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Abstract

Freeze‐fracture is a unique investigative tool for visualization of the en face topography of individual membrane leaflets of cell membranes at high resolution under the electron microscope. The development of a system of freeze‐fracture cytochemical and immunocytochemical techniques has further advanced the utility of this methodological approach for high‐resolution localization of specific membrane and intracellular macromolecules in tissues and cells. The unit focuses on description, in a step‐by‐step manner, of the experimental procedures for two specific freeze‐fracture labeling techniques, namely fracture‐label and label‐fracture. Users are guided in a stepwise manner, starting from the preparation of tissue or cell samples to the final retrieval and mounting of fracture‐label and label‐fracture specimens for examination on the electron microscope. © 2014 by John Wiley & Sons, Inc.

Keywords: freeze‐fracture immunocytochemistry; cell membranes; immunolabeling; fracture‐label; label‐fracture

     
 
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Table of Contents

  • Introduction
  • Fracture‐Label
  • Basic Protocol 1: Critical‐Point Drying (CPD) Fracture‐Label
  • Basic Protocol 2: Thin‐Section Fracture‐Label
  • Basic Protocol 3: Label‐Fracture
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Critical‐Point Drying (CPD) Fracture‐Label

  Materials
  • Tissue samples (which can be of any type of tissues either from humans or animals)
  • 2.5% and 1% (v/v) glutaraldehyde in PBS, pH 7.4
  • Phosphate‐buffered saline (PBS), pH 7.4 ( )
  • 30% (w/v) bovine serum albumin (BSA) in 0.1 M PBS (see for PBS)
  • Glutaraldehyde
  • 30% (v/v) glycerol in PBS
  • Liquid nitrogen
  • Propane
  • Crushed dry ice
  • 1% (v/v) glutaraldehyde in PBS containing 30% (v/v) glycerol
  • 1 mM glycylglycine in PBS containing 3% (v/v) glycerol
  • Probe: e.g., antibody
  • Gold‐conjugated secondary antibody or protein A–gold complex (see unit )
  • 1% (w/v) osmium tetroxide in distilled H 2O
  • 30%, 50%, 70%, 80%, 90%, and 100% (v/v) ethanol
  • Platinum inserts (Balzers AG, cat no. BD481505)
  • Carbon rods (Balzers AG, cat no. BD484055)
  • Sodium hypochlorite solution (bleach)
  • 12‐mm × 75‐mm (5‐ml) round‐bottom polypropylene tubes
  • 1.2‐ or 2.0‐ml cryogenic vials (e.g., Nalgene)
  • Styrofoam bucket
  • 10‐cm glass petri dish
  • 5‐in. stainless steel scalpel
  • Stainless steel forceps
  • Critical‐point dryer with carbon dioxide supply (SPI‐DRY CPD; SPI Supplies/Structure Probe)
  • Epon embedding BEEM capsules (Polysciences, Inc.)
  • Dissecting microscope
  • Balzers‐type specimen support plate or specimen transfer table (Balzers AG, cat no. BB172221‐T) coated with a strip of double‐faced adhesive tape
  • Freeze‐fracture unit (e.g., Balzers BAF400 Freeze Etching System)
  • Porcelain spot plate
  • Fine tweezers
  • Formvar‐coated copper grids (200 mesh; Electron Microsopy Sciences)
  • Filter paper
  • Additional reagents and equipment for cryo‐immunogold labeling (unit )

Basic Protocol 2: Thin‐Section Fracture‐Label

  Materials
  • Tissue samples (which can be of any type of tissues either from humans or animals)
  • Liquid nitrogen
  • 30% (v/v) glycerol in PBS
  • 1% (v/v) glutaraldehyde in PBS containing 30% (v/v) glycerol
  • 1 mM glycylglycine in PBS containing 3% (v/v) glycerol
  • Phosphate‐buffered saline (PBS; )
  • Probe: e.g., antibody
  • Gold‐conjugated secondary antibody or protein A–gold complex (see unit )
  • 1% (w/v) osmium tetroxide in distilled H 2O
  • 30%, 50%, 70%, 80%, 90%, and 100% (v/v) acetone
  • Epon embedding kit (Sigma‐Aldrich, cat. no. 45359‐1EA‐F)
  • 15‐ml Dounce tissue grinder set with a tissue grinder tube and a glass pestle
  • 12‐mm × 75‐mm polypropylene tubes with a round bottom
  • Epon embedding BEEM capsules (Polysciences, Inc.)
  • Centrifuge
  • 60°C oven
  • Formvar‐coated copper grids (200 mesh)
  • Additional reagents and equipment for preparing frozen BSA gel blocks for freeze‐fracture ( protocol 1, steps 1 to 7) and cryo‐immunogold labeling (unit )

Basic Protocol 3: Label‐Fracture

  Materials
  • Samples (which can be of any isolated cells, cells in a monolayer, or isolated membrane fractions)
  • Fixative of choice: e.g., 2.5% glutaraldehyde in PBS, pH 7.4
  • 0.1 M glycine in PBS
  • Probe: e.g., antibody
  • Gold‐conjugated secondary antibody or protein A–gold complex (see unit )
  • Phosphate‐buffered saline (PBS; )
  • 30% (v/v) glycerol in PBS
  • Liquid nitrogen
  • Propane
  • Centrifuge
  • Drawn Pasteur pipets
  • Balzers‐type specimen support plates (Balzers AG, cat. no. BB187280)
  • Pair of centering tweezers (Balzers AG, cat. no. BB801012038)
  • Double replica specimen table (Balzers AG, cat. no. BB17222)
  • Freeze‐fracture unit (e.g., Balzers BAF400 Freeze‐Etching System)
  • Spot plate
  • Formvar‐coated copper grids (200 mesh)
  • Additional reagents and equipment for cryo‐immunogold labeling (unit )
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Figures

Videos

Literature Cited

Literature Cited
   Forsman, C.A. and Pinto da Silva, P. 1988. Fracture‐flip: New high‐resolution images of cell surfaces after carbon stabilization of freeze‐fractured membranes. J. Cell Sci. 90:531‐541.
   Fujimoto, K. 1995. Freeze‐fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins: Application to the immunogold labeling of intercellular junctional complexes. J. Cell Sci. 108:3443‐3449.
   Fujimoto, K. 1997. SDS‐digested freeze‐fracture replica labeling electron microscopy to study the two‐dimensional distribution of integral membrane proteins and phospholipids in biomembranes: Practical procedure, interpretation and application. Histochem. Cell Biol. 107:87‐96.
   Gathercole, D.V. , Colling, D.J. , Skepper, J.N. , Takagishi, Y. , Levi, A.J. , and Severs, N.J. 2000. Immunogold‐labeled L‐type calcium channels are clustered in the surface plasma membrane overlying junctional sarcoplasmic reticulum in guinea‐pig myocytes: Implications for excitation‐contraction coupling in cardiac muscle. J. Mol. Cell. Cardiol. 32:1981‐1994.
   Hisano, S. and Daikoku, S. 1993. Colloidal gold methods. In Electron Microscopic Cytochemistry and Immunocytochemistry in Biomedicine ( K. Ogawa and T. Barka , eds.) pp. 303‐308. CRC Press, Boca Raton, Fla.
   Kan, F.W.K. 1993. Cytochemical evidence for the presence of phospholipids in epithelial junctional strands. J. Histochem. Cytochem. 41:649‐656.
   Kan, F.W.K. 1995. Backscattered electron imaging of colloidal gold‐labeled fracture‐label preparations. In Rapid Freezing, Freeze Fracture and Deep Etching ( N.J. Severs and D.M. Shotton , eds.) pp. 209‐224. Wiley‐Liss, New York.
   Kan, F.W.K. and Lin, Y. 1996. Immunogold localization of actin in the testis and exocrine pancreas: Spatial relationship with tight junctional strands. Microsc. Res. Tech. 31:128‐140.
   Kan, F.W.K. and Pinto da Silva, P. 1986. Preferential association of glycoproteins to the euchromatin regions is revealed by fracture‐label of cross‐fractured nuclei. J. Cell Biol. 102:576‐587.
   Kan, F.W.K. and Pinto da Silva, P. 1987. Molecular demarcation of surface domains as established by label‐fracture cytochemistry of boar spermatozoa. J. Histochem. Cytochem. 35:1069‐1078.
   Kan, F.W.K. and Pinto da Silva, P. 1989. Label‐fracture cytochemistry. In Vol. II: Colloidal Gold: Methods and Applications ( M. Hayat , ed.) pp. 175‐201. Academic Press, Orlando and London.
   Moor, H. , Mühlethaler, K. , Waldner, H. , and Frey‐Wyssling, A. 1961. A new freezing‐ultramicrotome. J. Biophys. Biochem. Cytol. 10:1‐13.
   Pavan, A. , Mancini, P. , Frati, L. , Torrisi, M.R. , and Pinto da Silva, P. 1989. Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label‐fracture and by fracture‐label. Biochim. Biophys. Acta 878:158‐168.
   Pinto da Silva, P. and Kan, F.W.K. 1984. Label‐fracture: A method for high resolution labeling of cell surfaces. J. Cell Biol. 99:1156‐1161.
   Pinto da Silva, P. , Kachar, B. , Torrisi, M.R. , Brown, C. , and Parkison, C. 1981a. Freeze‐ fracture cytochemistry: Replica of critical point‐dried cells and tissues after fracture‐label. Science 213:230‐233.
   Pinto da Silva, P. , Parkison, C. , and Dwyer, N. 1981b. Freeze‐fracture cytochemistry: Thin sections of cells and tissues after labeling of fracture faces. J. Histochem. Cytochem. 29:917‐928.
   Robenek, H. and Severs, N.J. 2008. Recent advances in freeze‐fracture electron microscopy: The replica immunolabeling technique. Biol. Proceed. Online 10:9‐19.
   Severs, N.J. 1995. Freeze‐fracture cytochemistry: An explanatory survey of methods. In Rapid Freezing, Freeze Fracture, and Deep Etching ( N.J. Severs and D.M. Shotton , eds.) pp. 173‐208. Wiley‐Liss, New York.
   Severs, N.J. 2007. Freeze‐fracture electron microscopy. Nat. Protoc. 2:547‐576.
   Severs, N.J. and Robenek, H. 2008. Freeze‐fracture cytochemistry in cell biology. Methods Cell Biol. 88:181‐204.
   Slot, J.W. and Geuze, H.J. 1985. A new method of preparing gold probes for multiple‐labeling cytochemistry. Eur. J. Cell Biol. 38:87‐93.
   Sorice, M. , Garofalo, T. , Misasi, R. , Dolo, V. , Lucania, G. , Sansolini, T. , Parolini, I. , Sargiacomo, M. , Torrisi, M.R. , and Pavan, A. 1999. Glycosphingolipid domains on cell plasma membrane. Bios. Rep. 19:197‐208.
   Squarzoni, S. , Sabatelli, P. , Maltarello, M.C. , Cataldi, A. , Di Primio, R. , and Maraldi, N.M. 1992. Localization of dystrophin COOH‐terminal domain by the fracture‐label technique. J. Cell Biol. 118:1401‐1409.
   Steere, R.L. 1957. Electron microscopy of structural detail in frozen biological specimens. J. Biophys. Biochem. Cytol. 3:45‐60.
   Torrisi, M.R. , Di Lazzaro, C. , Pavan, A. , Pereira, L. , and Campadelli‐Fiume, G. 1992. Herpes simplex virus envelopment and maturation studied by fracture‐label. J. Virol. 66:554‐561.
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