Cryo‐Immunogold Electron Microscopy

Peter J. Peters1, Erik Bos1, Alexander Griekspoor1

1 Netherlands Cancer Institute, Amsterdam, null
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.7
DOI:  10.1002/0471143030.cb0407s30
Online Posting Date:  April, 2006
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This unit describes subcellular localization of proteins/antigens using high‐resolution cryo‐immunogold electron microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Support protocols describe how to embed fixed material in gelatin, cryosection, and mount the sections on Formvar‐coated grids. This unit is accompanied by eleven videos that illustrate many of the procedures used in this unit.

Keywords: topological biochemistry; electron microscopy; subcellular localization; immunodetection; cryo‐immunogold electron microscopy

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Table of Contents

  • Introduction
  • Basic Protocol 1: Immunogold Labeling
  • Alternate Protocol 1: Immunogold Labeling with Silver Enhancement
  • Support Protocol 1: Aldehyde Fixation of Cells for Cryo‐Immunogold Labeling
  • Support Protocol 2: Fixation of Tissue for Cryo‐Immunogold Labeling
  • Support Protocol 3: Embedding Samples for Cryo‐Immunogold Labeling
  • Support Protocol 4: Cryosectioning for Cryo‐Immunogold Labeling
  • Support Protocol 5: Preparation of Carbon‐ and Formvar‐Coated Copper Grids
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Immunogold Labeling

  • Cells or tissue of interest, fixed ( protocol 3 or protocol 42), embedded ( protocol 5), cryo‐sectioned ( protocol 6), and mounted on Formvar‐coated grids ( protocol 7)
  • 2% (w/v) gelatin‐coated 2.5‐cm Petri dishes
  • PBS ( appendix 2A) containing 0.15 M glycine
  • PBS containing 1% (w/v) BSA
  • Primary antibody diluted in PBS/1% BSA
  • PBS/0.1% (w/v) BSA
  • Secondary antibody in PBS/1% BSA (optional)
  • 10‐nm protein A–gold particles at OD 0.1 (commercially available from Cell Biology, Medical School, Utrecht University, The Netherlands) in PBS/1% BSA
  • PBS containing 1% (v/v) glutaraldehyde
  • Uranyl oxalate solution (see recipe; optional)
  • Methyl cellulose/uranyl acetate solution (see recipe)
  • Parafilm M
  • Whatman no. 50 filter paper
  • 37°C incubator
  • Forceps
  • Stainless steel loop slightly larger than grids attached to P1000 (blue, 1000‐µl) pipet tips (see Video 1 at

Alternate Protocol 1: Immunogold Labeling with Silver Enhancement

  • Gelatin storage plates containing grids with cryosections
  • PBS containing 0.5% (w/v) cold fish skin gelatin (CFG)
  • Fab fragments of the primary antibody conjugated to Aurion ultrasmall gold particles
  • Ultrasmall gold silver enhancement kit (Aurion)

Support Protocol 1: Aldehyde Fixation of Cells for Cryo‐Immunogold Labeling

  • Cultures of adherent cells (from a 10‐cm dish) or cells in suspension
  • Culture medium appropriate to the cell type
  • 2× fixative, pre‐warmed to 37°C: 2× FA (see recipe) or 2× FA/GA (see recipe)
  • Storage solution (see recipe)
  • PBS containing 0.15 M glycine, 37°C
  • PBS containing 1% (w/v) gelatin
  • Cell scraper
  • Glass Pasteur pipets (do not use plastic since several cell types may adhere to plastic)
  • Table‐top centrifuge
  • 1.5‐ml microcentrifuge tubes
  • 15‐ml tubes (Falcon)

Support Protocol 2: Fixation of Tissue for Cryo‐Immunogold Labeling

  • Tissue of interest
  • 1x fixative: 1× FA (see recipe), or 1× FA/GA (see recipe)
  • Storage solution (see recipe)
  • Razor blades, acetone cleaned
  • Glass vials with screw caps
  • Fine forceps

Support Protocol 3: Embedding Samples for Cryo‐Immunogold Labeling

  • Fixed cells (see protocol 3) or tissue (see protocol 4) in storage solution
  • PBS
  • PBS containing 0.15 M glycine
  • 12% (w/v) gelatin in 0.1 M sodium phosphate buffer (see recipe), 37°C
  • 2% and 5% (w/v) gelatin in 0.1 M sodium phosphate buffer (see recipe), 37°C (for tissue)
  • 2.3 M sucrose in 0.1 M sodium phosphate buffer, pH 7.4
  • Parafilm M
  • Glass Pasteur pipets
  • Razor blades, acetone‐rinsed and air‐dried
  • Dissecting microscope with cold‐light optics
  • 1‐ml plastic vials
  • 10‐ml vials
  • Table‐top centrifuge
  • End‐over‐end rotator (∼10 sec per rotation)
  • Fine forceps
  • Greiner analyzer cup 14/24‐mm, 1.5‐ml, and caps (Greiner Bio‐One)
  • Aluminum specimen holders, or steel pins, roughened with sandpaper and acetone‐cleaned, dust‐free
  • 15‐ml aluminum cryo tubes with two holes punched in the top
  • Liquid nitrogen

Support Protocol 4: Cryosectioning for Cryo‐Immunogold Labeling

  • Sample blocks that are stored in liquid nitrogen (see protocol 5)
  • Section retrieval solution (see recipe), freshly prepared
  • Ultramicrotome (Leica) with cryochamber and antistatic devices (Diatome)
  • Diamond knife (Diatome; cryo‐immuno 35°)
  • Trimming knife (Diatome; cryo‐trim 45° or 20°)
  • Eyelash; undamaged, clean and mounted on a wooden stick (see Video 7 at
  • 3‐mm diameter loop made of twisted 0.3‐mm romanium wire (Winkelstroeter Dentaurum) on a 15‐cm wooden stick (see Video 1)
  • Carbon‐ and formvar‐coated copper grids (see protocol 7)

Support Protocol 5: Preparation of Carbon‐ and Formvar‐Coated Copper Grids

  • 6% (w/v) ammonium hydroxide solution
  • Acetone
  • Formvar (Merck)
  • Chloroform, analytical grade
  • 100‐mesh copper grids or one‐hole grids
  • 100‐ml glass‐stoppered Erlenmeyer flask
  • Glass microscope slides
  • Coplin jar
  • Adhesive paper, e.g., address labels for envelopes
  • Formvar/carbon‐coating device (BOC Edwards or equivalent)
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  •   FigureFigure 4.7.1 Light microscope and electron microscope micrographs of two adjacent ultrathin cryosection of a dentritic cell. LM MHC class II was detected together with CD1b by immunofluorescence on semithin cryosections of 280 nm. The CD1b is presented in the upper left panel; MHC class II in the lower left panel and a merge of the pictures is shown in the left bottom panel. CD1b and MHC class II molecules are located intracellular in the lysosomal MHC class II compartments (MIIC; see Peters et al., ). Note that details of immunofluorescence pattern is comparable if not better then confocal laser scan microscopy. EM micrograph (right panel) of a 50‐nm section. Immunogold labeling of CD1b (small gold) and MHC class II (large gold) within MIIC. Please note that by EM one can see a differential distribution of CD1b versus MHC class II within the MIIC not seen by LM. The CD1b molecules reside on the outer membrane while class II molecules are on the internal membranes of the MIIC.
  •   FigureFigure 4.7.2 LM Hippocampal semithin cryosections labeled for PrPC with Fab D18. Labeling is concentrated in the stratum oriens (o), stratum radiatum (r) and lacunosum‐moleculare (lm); cell bodies of pyramidal (p) and granule (g) layers are free of labeling with the exception of a few cells. Upper left: Fab D18 was used with a secondary polyclonal antibody and protein A–gold (5 nm) that was subsequently visualized by silver enhancement (Aurion). Upper right: Fluorescent labeling shows a labeling pattern similar to panel A. EM PrP is concentrated in the cytosol in a subpopulation of neurons. Lower left: protein–A gold particles label PrP. Lower right: Direct immunolabeling with Fab D18‐ultrasmall gold (Aurion) and silver enhancement demonstrates the same cytPrP abundance.

Literature Cited

   Aebi, U. and Pollard, T.D. 1987. A glow discharge unit to render microscope grids and other surfaces hydrophilic. J. Electron Microsc. Tech. 7:29‐33.
   Batten, T.F. and Hopkins, C.R. 1979. Use of protein A‐coated colloidal gold particles for immuno‐electronmicroscopic localization of ACTH on ultrathin sections. Histochemistry 60:317‐220.
   Christensen, A.K. 1971. Frozen thin sections of fresh tissue for electron microscopy, with a description of pancreas and liver. J. Cell Biol. 51:772‐804.
   Griffiths, G. and Posthuma, J. 2002. A reliable and convenient method to store ultrathin thawed cryoscetions prior to immunolabeling. J. Histochem. Cytochem. 50:57‐62.
   Griffiths, G., Brands, R., Burke, B., Louvard, D., and Warren, G. 1982. Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport. J. Cell Biol. 95:781‐792.
   Lefman, J., Zhang, P., Hirai, T., Juliani, J., Bliss, D., Kessel, M., Bos, E., Weis, R., Peters, P.J., and Subramaniam, S. 2004. Three‐dimensional imaging of chemotaxis receptor networks in a bacterial cell. J. Bacteriol. 186:5052‐5061.
   Liou, W., Geuze, H.J., and Slot, J.W. 1996. Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106:41‐58.
   Mayhew, T.M., Lucocq, J.M., and Griffiths, G. 2002. Relative labeling index: A novel stereological approach to test for non‐random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections. J. Microsc. 205:153‐164.
   Mironov, A. Jr., Latawiec, D., Wille, H., Bouzamondo‐Bernstein, E., Legname, G., Williamson, R.A., Burton, D., DeArmond, S.J., Prusiner, S.B., and Peters, P.J. 2003 Cytosolic prion protein in neurons. J. Neurosci. 23:7183‐7193.
   Peters, P.J. and Hunziker, W. 2001. Subcellular localization of Rab17 by cryo‐immunogold electron microscopy in epithelial cells grown on polycarbonate filters. Methods Enzymol. 329:210‐225.
   Peters, P.J., Neefjes, J.J., Oorschot, V., Ploegh, H.L., and Geuze, H.J. 1991a. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature 349:669‐676.
   Peters, P.J., Borst, J., Oorschot, V., Fukuda, M., Krahenbuhl, O., Tschopp, J., Slot, J.W., and Geuze, H.J. 1991b. Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J. Exp. Med. 173:1099‐1109.
   Peters, P.J., Mironov, A. Jr., Peretz, D., van Donselaar, E., Leclerc, E., Erpel, S., DeArmond, S.J., Burton, D.R., Williamson, R.A., Vey, M., and Prusiner, S.B. 2003. Trafficking of prion proteins through a caveolae‐mediated endosomal pathway. J. Cell Biol. 162:703‐717.
   Raposo, G., Kleijmeer, M.J., Posthuma, G., Slot, J.W., and Geuze, H.J. 1997. Immunogold labeling of ultrathin cryosections: Application in immunology. In Handbook of Experimental Immunology. Vol 4, 5th ed. (M.A. Cambridge, L.A. Herzenberg, D. Weir, L.A. Herzenberg, and C. Blackwell, eds.) pp. 208:1‐11. Blackwell Scientific, Oxford.
   Romano, E.L. and Romano, M. 1977. Staphylococcal protein A bound to colloidal gold: A useful reagent to label antigen‐antibody sites in electron microscopy. Immunochemistry 14:711‐715.
   Roth, J. 1996. The silver anniversary of gold: 25 years of the colloidal gold marker system for immunocytochemistry and histochemistry. Histochem. Cell Biol. 106:1‐8.
   Roth, J. and Berger, E.G., 1982. Immunocytochemical localization of galactosyltransferase in HeLa cells: Codistribution with thiamine pyrophosphatase in trans Golgi cisternae. J. Cell Biol. 93:223‐229.
   Roth, J., Bendayan, M., and Orci, L. 1978. Ultrastructural localization of intracellular antigens by the use of protein A‐gold complex. J. Histochem. Cytochem. 26:1074‐1081.
   Roth, J., Thorens, B., Hunziker, W., Norman, A.W., and Orci, L. 1981. Vitamin D–dependent calcium binding protein: Immunocytochemical localization in chick kidney. Science 214:197‐200.
   Slot, J.W. and Geuze, H.J. 1981. Sizing of protein A‐colloidal gold probes for immunoelectron microscopy. J. Cell Biol. 90:533‐536.
   Slot, J.W., Geuze, H.J., Gigengack, S., Lienhard, G.E., and James, D.E. 1991. Immuno‐localization of the insulin regulatable glucose transport in brown adipose tissue of rat. J. Cell Biol. 113:123‐135.
   Stoorvogel, W., Oorschot, V., and Geuze, H.J. 1996. A novel class of clarithin‐coated vesicles budding from endosomes. J. Cell. Biol. 132:21–33.
   Swanson, J.A. and Peters, P.J. 2005. Subcellular imaging techniques—microscopic visual imaging. Curr. Opin. Microbiol. 8:313‐315.
   Tokuyasu, K.T. 1973. A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57:551‐565.
   Tokuyasu, K.T. 1978. A study of positive staining of ultrathin frozen sections. J. Ultrstruct. Res 63:287‐307.
   Tokuyasu, K.T. and Singer, S.J. 1976. Improved procedures for immunoferritin of ultrathin frozen sections. J. Cell Biol. 71:894‐906.
   Touret, N., Paroutis, P., Terebiznik, S., Pypaert, M., Chow, A., Jiang, A., Ghaio, J., Yip, C., Moore, H., van der Wel, N., Houben, D., Peters, P., de Chastellier, C., Mellman, I., and Grinstein, S. 2005. Quantitative and dynamic assessment of the contribution of the endoplasmic reticulum to phagosome formation. Curr. Opin. Microbiol. 8:151‐170.
   van der Wel, N.N., Sugita, M., Fluitsma, D.M., Cao, X., Schreibelt, G., Brenner, M.B., and Peters, P.J. 2003. CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation. Mol. Biol. Cell 14:3378‐3388.
   van der Wel, N.N., Fluitsma, D.M., Dascher, C.C., Brenner, M.B., and Peters, P.J., 2005. Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo‐techniques for light and electron microscopy. Curr. Opin. Microbiol. 8:323‐330.
Internet Resources
  The website for more information about the methods described in this unit and related techniques. For more information contact Peter J. Peters at
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