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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Abstract

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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Materials

Basic Protocol 1: Immunogold Labeling

  Materials
  • 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 http://www.currentprotocols.com).

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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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 http://www.currentprotocols.com).
  • 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

  Materials
  • 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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Figures

  •   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

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Internet Resources
   http://www.nki.nl/nkidep/h4/peters/peters.htm
  The website for more information about the methods described in this unit and related techniques. For more information contact Peter J. Peters at p.peters@nki.nl.
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