Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues

William J. Brown1

1 Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.6
DOI:  10.1002/0471143030.cb0406s01
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This carefully written unit describes two methods for using the immunoperoxidase reaction to localize antigens at the electron microscope level; one for adherent cultured cells and one for tissue sections. The reaction conditions are first optimized at the light microscope level and then adapted for EM level observation. These methods allow for reliable detection of antigens at the cell surface, within the cell, and especially in membrane bounded organelles. Embedding and staining procedures are also optimized for sample visualization.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Immunoperoxidase Staining of Cultured Cells
  • Alternate Protocol 1: Detachment and Embedding of Cultured Cells for Analysis of ER Antigens
  • Alternate Protocol 2: Embedding of Cultured Cells for Analysis of Monolayer Sections
  • Basic Protocol 2: Immunoperoxidase Staining of Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Immunoperoxidase Staining of Cultured Cells

  Materials
  • Cells of interest
  • PLP fixative (see recipe), freshly prepared
  • PBS, pH 7.4 ( appendix 2A)
  • 0.005% to 0.05% (w/v) saponin (Sigma)/PBS
  • Primary antibody in saponin/PBS with 0.1% (w/v) ovalbumin
  • HRP‐conjugated secondary antibody: horseradish peroxidase–conjugated Fab fragments of anti–rabbit IgG or anti–mouse IgG (Biosys) in saponin/PBS with 0.1% (w/v) ovalbumin
  • Glutaraldehyde fixative: 1.5% (v/v) glutaraldehyde in 5% (w/v) sucrose/100 mM sodium cacodylate, pH 7.4
  • 7.5% (w/v) sucrose/100 mM sodium cacodylate, pH 7.4
  • 0.2% (w/v) DAB solution (see recipe), freshly prepared
  • 3% (v/v) H 2O 2, freshly prepared from 30% H 2O 2 stock solution
  • 1% (w/v) reduced OsO 4 solution (see recipe), freshly prepared and ice cold
  • 100 mM sodium cacodylate, pH 7.4, ice cold
  • 70%, 95%, and 100% (v/v) ethanol
  • 100% propylene oxide, EM grade
  • 1:1 (v/v) propylene oxide/plastic embedding resin (e.g., Spurr's)
  • 100% plastic embedding resin (e.g., Spurr's)
  • 35‐mm disposable plastic petri dishes
  • Horizontal reciprocating shaker
NOTE: The propylene oxide detachment method of embedding can be somewhat tricky. It is advisable to practice on cells before attempting the immunoperoxidase staining (see step ).

Alternate Protocol 1: Detachment and Embedding of Cultured Cells for Analysis of ER Antigens

  • Epon 812 resin
  • Embedding molds

Alternate Protocol 2: Embedding of Cultured Cells for Analysis of Monolayer Sections

  Materials
  • Animal tissue of interest
  • PLP fixative (see recipe), freshly prepared
  • PBS, pH 7.4 ( appendix 2A)
  • 10% (v/v) dimethyl sulfoxide (DMSO) in PBS, 4°C
  • Isopentane (2‐methylbutane)
  • Liquid nitrogen
  • Tissue‐Tek OCT compound (Baxter)
  • 0.1% (w/v) ovalbumin/PBS
  • Primary antibody in 0.1% ovalbumin/PBS with 0.02% (w/v) sodium azide (NaN 3)
  • HRP‐conjugated secondary antibody: horseradish peroxidase–conjugated Fab fragments of anti–rabbit IgG or anti–mouse IgG (Biosys) in 0.1% ovalbumin/PBS
  • Glutaraldehyde fixative: 1.5% (v/v) glutaraldehyde in 5% (w/v) sucrose/100 mM sodium cacodylate, pH 7.4
  • 7.5% (w/v) sucrose/100 mM sodium cacodylate, pH 7.4
  • 0.2% (w/v) DAB solution (see recipe), freshly prepared
  • 3% (v/v) H 2O 2, freshly prepared from 30% H 2O 2 stock solution
  • 1% (w/v) reduced OsO 4 solution (see recipe), freshly prepared and ice cold
  • 100 mM sodium cacodylate, pH 7.4, ice cold
  • 70%, 95%, and 100% (v/v) ethanol
  • 100% propylene oxide, EM grade
  • 1:1 (v/v) propylene oxide/plastic embedding resin (e.g., Spurr's)
  • 100% plastic embedding resin (e.g., Spurr's)
  • Large styrofoam box or container
  • Cryostat‐type tissue sectioner
  • 10 × 75–mm test tubes
  • Slow‐moving shaker or mixer
  • Flat embedding molds
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 4.6.1 Immunoperoxidase localization of resident Golgi‐complex enzymes in cultured cells and cells from tissue sections. (A and B) Rat clone 9 hepatocytes stained by the methods described in for cultured cells with antibodies to the medial Golgi enzyme α‐mannosidase II. In (A), the very electron‐dense DAB reaction product (arrow) stands out in sharp contrast to the rest of the cytoplasm and fills mainly one or two cisternal elements of the Golgi stack. This example illustrates a well‐preserved, immunolabeled cell whose reacted organelle membranes are morphologically intact. Note that the DAB reaction product remains sequestered within the lumen of the cisternae. In (B), another clone 9 cell, reacted with the same antibody, shows significant damage resulting from cisternal elements that have become extremely dilated (“exploded”) and broken (arrowheads). As a consequence, the DAB reaction product leaks into the cytoplasm (arrow). In (C), a portion of a rat pancreatic acinar cell is shown. The cell was labeled with antibodies against sialyltransferase, a trans Golgi enzyme, by the methods described in . In this cell, the DAB reaction product also stands in sharp contrast to the cytoplasm and is sequestered within an intact cisternae (arrow). Abbreviations: cv, condensing vacuole; Gc, Golgi cisternae; n, nucleus; rer, rough endoplasmic reticulum. Scale bars, 0.5 µm.

Videos

Literature Cited

Literature Cited
   Broadwell, R.D., Oliver, C., and Brightman, M.W. 1979. Localization of neurophysin within organelles associated with protein synthesis and packaging in the hypothalamoneurophysial system: An immunocytochemical study. Proc. Natl. Acad. Sci. U.S.A. 76:5999‐6003.
   Brown, W.J. and Farquhar, M.G. 1984. The mannose‐6‐phosphate receptor for lysosomal enzymes is localized in the cis Golgi cisternae. Cell 36:295‐307.
   Brown, W.J. and Farquhar, M.G. 1989. Immunoperoxidase methods for the localization of antigens in cultured cells and tissue sections by electron microscopy. Methods Cell Biol. 31:553‐569.
   Brown, W.J., Constantinescu, E., and Farquhar, M.G. 1984. Redistribution of mannose‐6 phosphate receptors induced by tunicamycin and chloroquine. J. Cell Biol. 99:320‐326.
   Courtoy, P.J., Kanwar, Y.S., Hynes, R.O., and Farquhar, M.G. 1980. Fibronectin localization in the rat glomerulus. J. Cell Biol. 87:691‐696.
   Courtoy, P.J., Timpl, R., and Farquhar, M.G. 1982. Comparative distribution of laminin, type IV collagen, and fibronectin in the rat glomerulus. J. Histochem. Cytochem. 30: 874‐886.
   Courtoy, P.J., Picton, D.H., and Farquhar, M.G. 1983. Resolution and limitations of the immunoperoxidase procedure in the localization of extracellular matrix antigens. J. Histochem. Cytochem. 31:945‐951.
   Graham, R.C. and Karnovsky, M.J. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14:291‐302.
   Karnovsky, M.J. 1971. Use of ferrocyanide‐reduced osmium tetroxide in electron microscopy. J. Cell Biol. 51:146a.
   Kerjaschki, D. and Farquhar, M.G. 1983. Immunocytochemical localization of the Heyman's nephritis antigen (gp330) in glomerular epithelial cells of normal Lewis rats. J. Exp. Med. 157:667‐686.
   Li, J.Y., De Camilli, P., and Dahlstrom, A. 1997. Intraneuronal trafficking and distribution of amphiphysin and synaptojanin in the rat peripheral nervous system and the spinal cord. Eur. J. Neurosci. 9:1864‐1874.
   Louvard, D., Reggio, H., and Warren, G. 1982. Antibodies to the Golgi complex and the rough endoplasmic reticulum J. Cell Biol. 92:92‐107.
   McLean, I.W. and Nakane, P.K. 1974. Periodate‐lysine‐paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22:1077‐1083.
   Norgren, R.B. and Lehman, M.N. 1989. A double‐label pre‐embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers. J. Histochem. Cytochem. 37:1283‐1289.
   Novikoff, A.B. 1973. Studies on the structure and function of cell organelles: 3,3′‐Diaminobenzidine cytochemistry. In Electron Microscopy and Cytochemistry (E., Wisse, W., Daems, I., Molenaar, and P., Van Duijn, eds.) pp. 89‐109. North‐Holland Publishing, New York.
   Novikoff, A.B. 1980. DAB cytochemistry: Artifact problems in its current use. J. Histochem. Cytochem. 28:1036‐1038.
   Novikoff, A.B. and Goldfischer, S., 1969. Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem. 17:675‐680.
   Novikoff, A.B., Novikoff, P.M., Stockert, R.J., Becker, F.F., Yam, A., Poruchynsky, M.S., Levin, W., and Thomas, P.E. 1979. Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2‐acetylaminofluorene. Proc. Natl. Acad. Sci. U.S.A. 76:5207‐5211.
   Saraste, J., Lahtinen, U., and Goud, B. 1995. Localization of the small GTP‐binding protein rab1p to early compartments of the secretory pathway. J. Cell Sci. 108:1541‐1552.
   Todd, A.J. 1997. A method for combining confocal and electron microscopic examination of sections processed for double or triple labelling immunocytochemistry. J. Neurosci. Methods 73:149‐157.
   Tougard, C., Picart, R., and Tixier‐Vidal, A. 1980. Electron microscopic studies on the secretory process in rat prolactin cells in primary culture. Am. J. Anat. 158:471‐490.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library