The Importance of Titrating Antibodies for Immunocytochemical Methods

Gloria E. Hoffman1, Wei Wei Le1, Luciane V. Sita2

1 Department of Anatomy and Neurobiology, University of Maryland, Baltimore, Maryland, 2 Department of Anatomy, University of São Paulo, São Paulo, Brazil
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.12
DOI:  10.1002/0471142301.ns0212s45
Online Posting Date:  October, 2008
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Abstract

When using immunocytochemistry, investigators may not know how to optimize staining or how to troubleshoot the method when staining fails. Lacking are guides for comparing techniques and applying information derived from one staining method to another. Newer methods amplify signal detection, but will not necessarily work at the same primary antibody concentrations used for less sensitive reactions. Recommendations of optimal titers are often not accurate and are not usually accompanied by information on the method used to test those antibodies or the specifics of the assay. When the staining does not work, the investigators do not know how to determine if the antiserum is bad, the tissue is bad, or the method is inappropriate for their staining. This unit describes detailed procedures for determining optimal staining and applying that information to three common immunofluorescence methods. Lastly, a formula is provided for converting among the different methods. Curr. Protoc. Neurosci. 45:2.12.1‐2.12.26. © 2008 by John Wiley & Sons, Inc.

Keywords: immunoperoxidase; ABC technique; TSA amplification; immunofluorescence; immunocytochemistry; antibody dilution

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Titration of Antibodies using Immunocytochemistry
  • Alternate Protocol 1: Immunohistochemistry using Enzymatic Peroxide Generation with Glucose and a Glucose Oxidase Chromogen
  • Alternate Protocol 2: Fixation of Brain Tissue using Buffered 4% Paraformaldehyde without Acrolein
  • Alternate Protocol 3: Fixation of Brain Tissue using Borate Buffer (pH 9.5) without Acrolein
  • Alternate Protocol 4: Egg Yolk/Gelatin Embedding of Brain Tissue for Immunohistochemistry
  • Basic Protocol 2: Immunofluorescence Detection using Fluorophore‐Tagged Secondary Antibodies
  • Basic Protocol 3: Immunofluorescence using Biotin‐Tagged Secondary Antibodies and Streptavidin‐Conjugated Fluorophores
  • Basic Protocol 4: Biotinylated Tyramine Amplification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Titration of Antibodies using Immunocytochemistry

  Materials
  • Young adult Sprague‐Dawley rats
  • 1 U/µl heparin
  • 0.9% (w/v) NaCl containing 2% (w/v) sodium nitrite
  • 10% sodium bisulfite
  • 4% (w/v) paraformaldehyde containing 2.5% acrolein, in potassium phosphate buffer, pH 6.8 (see recipe)
  • 30% (w/v) sucrose, cold
  • Antifreeze cryoprotectant (see recipe)
  • 0.05 M KPBS (see recipe)
  • 0.1% (w/v) sodium borohydride in 0.05 M KPBS
  • 0.014% (w/v) phenylhydrazine hydrochloride or 1% (v/v) hydrogen peroxide in 0.05 M KPBS
  • Primary antibody
  • 0.05 M KPBS (see recipe) containing 0.4% (v/v) Triton X‐100
  • Biotinylated secondary antibody
  • Vectastain Elite ABC Kit (Standard; Vector Laboratories, cat. no. PK‐6100), including Solution A and Solution B
  • 0.175 M sodium acetate
  • NiDAB chromogen solution (see recipe)
  • 3% (v/v) H 2O 2
  • 50%, 70%, and 95% (v/v) ethanol
  • Absolute ethanol
  • Xylene or Histoclear (National Diagnostics)
  • Mounting medium: e.g., Permount (Fisher Scientific), Histomount (National Diagnostics), Krystalon (Harleco, cat. no. 64969, available from Voigt Global Distribution LLC, http://www.voigtglobal.com), or D.P.X. (Aldrich)
  • Surgical equipment
  • 15‐G needle
  • Peristaltic pump
  • Freezing sliding microtome or cryostat (also see unit 1.1)
  • Large petri dishes
  • Subbed glass slides (see recipe) or Fisher Superfrost electrostatically charged slides
  • Glass coverslips
  • Additional reagents and equipment for anesthesia of rodents ( appendix 4B) and sectioning of brain tissue (unit 1.1)
CAUTION: Whole‐animal perfusion should be performed in a hood with the animal on a dissecting tray or rack such that a “capture” tray can be placed below. The hood should be checked by the Institution's Health and Safety office to ensure proper air flow for use of acrolein.

Alternate Protocol 1: Immunohistochemistry using Enzymatic Peroxide Generation with Glucose and a Glucose Oxidase Chromogen

  • β‐D(+)‐glucose (C 6H 12O 6)
  • 3,3′‐diaminobenzidine tetrahydrochloride (DAB; Fluka, cat. no. 32750)
  • Nickel (II) sulfate hexahydrate (NiSO 4⋅6H 2O; Sigma, cat. no. N‐4882)
  • Sodium acetate–imidizole solution (see recipe)
  • 7.5 U/ml glucose oxidase (Sigma, cat. no. G‐0543)
Follow protocol 1, modifying only the chromogen solution in step 24.

Alternate Protocol 2: Fixation of Brain Tissue using Buffered 4% Paraformaldehyde without Acrolein

  • 4% paraformaldehyde in phosphate buffer pH 6.8 (see recipe for 4% paraformaldehyde plus 2.5% acrolein, but do not add the acrolein)

Alternate Protocol 3: Fixation of Brain Tissue using Borate Buffer (pH 9.5) without Acrolein

  • 0.9% (w/v) NaCl at 4°C (not room temperature)
  • 4% paraformaldehyde in borate buffer, pH 9.5 (see recipe), 4°C
  • 20% sucrose diluted in 4% paraformaldehyde in borate buffer, 4°C
  • 20% sucrose diluted in 0.05 M KPBS, 4°C
  • Normal serum from the same host as the biotinylated secondary antibody

Alternate Protocol 4: Egg Yolk/Gelatin Embedding of Brain Tissue for Immunohistochemistry

  • 12% and 6% gelatin (see recipe), prepared fresh
  • Fresh eggs at room temperature (remove from refrigerator several hours before use)
  • Peel‐A‐Way molds (Ted Pella, cat. no. 27116 for small tissues, or 27110 for larger samples)
  • 40°C water bath
  • Whatman no. 1 filter paper
  • Pin
  • Smooth‐tipped forceps

Basic Protocol 2: Immunofluorescence Detection using Fluorophore‐Tagged Secondary Antibodies

  Materials
  • Brain sections as obtained in protocol 1, steps 1 to 15
  • Primary antibody
  • Fluorophore‐conjugated secondary antibody
  • Additional reagents and equipment for titration of antibodies using immunocytochemistry ( protocol 1)

Basic Protocol 3: Immunofluorescence using Biotin‐Tagged Secondary Antibodies and Streptavidin‐Conjugated Fluorophores

  Materials
  • Brain sections as obtained in protocol 1, steps 1 to 15
  • Primary antibody
  • Biotinylated secondary antibody
  • Fluorophore‐conjugated streptavidin
  • Additional reagents and equipment for titration of antibodies using immunocytochemistry ( protocol 1)

Basic Protocol 4: Biotinylated Tyramine Amplification

  Materials
  • Brain sections as obtained in protocol 1, steps 1 to 15
  • Primary antibody
  • Biotinylated secondary antibody
  • Vectastain Elite ABC Kit (Standard; Vector Laboratories, cat. no. PK‐6100) including solution A and Solution B
  • Biotinylated tyramine: this reagent is included in TSA kits sold by Perkin Elmer.
  • 3% H 2O 2
  • Fluorophore‐conjugated streptavidin
  • Additional reagents and equipment for titration of antibodies using immunocytochemistry ( protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
   Adams, J.C. 1992. Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J. Histochem. Cytochem. 40:1457‐1463.
   Berghorn, K.A., Bonnett, J.H., and Hoffman, G.E. 1994. cFos immunoreactivity is enhanced with biotin amplification. J Histochem. Cytochem. 42:1635‐1642.
   Hoffman, G.E., Smith, M.S., and Fitzsimmons, M.D. 1992. Detecting steroidal effects on immediate early gene expression in the hypothalamus. Neuroprotocols 1:52‐66.
   Hsu, S.M., Raine, L. and Fanger, H. 1981. Use of avidin‐biotin‐peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem. Cytochem. 29:577‐580.
   RBI (Research Biochemicals International). 1998. Too much of a good thing. Neurotransmissions 14:14‐17.
   Sternberger, L., Hardy, P., Cuculus, J., and Meyer, H. 1970. The unlabeled antibody‐enzyme method of immunochemistry: Preparation and properties of soluble antigen‐antibody complex (horseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18:315‐333.
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