The Importance of Titrating Antibodies for Immunocytochemical Methods

Gloria E. Hoffman1, Kelley J. Murphy1, Luciane V. Sita2

1 Department of Biology, Morgan State University, Baltimore, Maryland, 2 Department of Anatomy, University of São Paulo, São Paulo
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.12
DOI:  10.1002/cpns.1
Online Posting Date:  July, 2016
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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. © 2016 by John Wiley & Sons, Inc.

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

     
 
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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
  • Basic Protocol 5: Immunohistochemistry Using the Pelco BioWave Pro Histological Microwave with Steadytemp Recirculating Water Bath
  • Support Protocol 1: Programming the BioWave Pro Histological Microwave
  • Basic Protocol 6: Microwave‐Assisted Immunofluorescence Detection Using Fluorophore‐Tagged Secondary Antibodies
  • Basic Protocol 7: Microwave‐Assisted Immunofluorescence Detection Using Biotin‐Tagged Secondary Antibodies and Streptavidin‐Conjugated Fluorophores
  • Basic Protocol 8: Microwave‐Assisted 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
  • Animal of choice
  • 1 U/μl heparin
  • 0.9% (w/v) NaCl containing 2% (w/v) sodium nitrite
  • 10% (w/v) sodium bisulfite
  • 4% (w/v) paraformaldehyde containing 2.5% (v/v) 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)
  • 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) hydrogen peroxide
  • 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), or DPX (Sigma 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 Plus electrostatically charged slides
  • Glass coverslips
  • Additional reagents and equipment for anesthesia of rodents ( appendix 4B) and sectioning of brain tissue (unit 1.1)
CAUTION: Acrolein is a potent volatile eye and respiratory irritant and must be handled with care. Whole‐animal perfusion and fixation should be performed in a fume hood with the animal on a dissecting tray or rack such that a “capture” tray can be placed below. The fume hood should be checked by the Institution's Health and Safety office to ensure proper air flow for use of acrolein. Availability of 10% sodium bisulfite solutions is important for neutralizing any acrolein that spills, escapes the animal, or is left over after perfusion.

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

  Additional Materials (also see protocol 1)
  • 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)

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

  Additional Materials (also see protocol 1)
  • 4% (w/v) 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

  Additional Materials (also see protocol 1)
  • 0.9% (w/v) NaCl at 4°C (not room temperature)
  • 4% (w/v) paraformaldehyde in borate buffer, pH 9.5 (see recipe), 4°C
  • 20% (w/v) sucrose diluted in 4% (w/v) paraformaldehyde in borate buffer, 4°C
  • 20% (w/v) 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

  Additional Materials (also see protocol 1)
  • 12% and 6% (w/v) 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 produced as outlined in Adams ( ) or purchased (e.g., ThermoFisher Scientific)
  • 3% (v/v) hydrogen peroxide
  • Fluorophore‐conjugated streptavidin
  • Additional reagents and equipment for titration of antibodies using immunocytochemistry ( protocol 1)

Basic Protocol 5: Immunohistochemistry Using the Pelco BioWave Pro Histological Microwave with Steadytemp Recirculating Water Bath

  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
  • NiDAB chromogen solution (see recipe)
  • BioWave Pro Laboratory Microwave System (Ted Pella Instruments, cat. no. 36500)
  • PELCO SteadyTemp Pro Digital Thermoelectric Recirculator (Ted Pella Instruments, cat. no. 50062)
  • 5‐ml plastic beaker cups (VWR, cat. no. 13915‐985)
  • Nets to fit inside beaker cups (Costar, cat. no. 3478)
  • Holder/tray for beaker cups
  • Transfer pipets (Fisher, cat. no. 13‐711‐7M)
  • Paintbrushes of various sizes
  • Additional reagents and equipment for titration of antibodies using immunocytochemistry ( protocol 1)

Support Protocol 1: Programming the BioWave Pro Histological Microwave

  Materials
  • Brain sections as obtained in protocol 1, steps 1 to 15
  • Primary antibody
  • Fluorophore‐conjugated secondary antibody
  • Additional reagents and equipment for microwave‐assisted immunocytochemistry ( protocol 9)

Basic Protocol 6: Microwave‐Assisted Immunofluorescence Detection Using Fluorophore‐Tagged Secondary Antibodies

  Materials
  • Brain sections as obtained in Basic Protocol, steps 1 to 15
  • Primary antibody
  • Biotinylated secondary antibody
  • Fluorophore‐conjugated streptavidin
  • Additional reagents and equipment for microwave‐assisted immunocytochemistry ( protocol 9)

Basic Protocol 7: Microwave‐Assisted Immunofluorescence Detection Using Biotin‐Tagged Secondary Antibodies and Streptavidin‐Conjugated Fluorophores

  Materials
  • Brain sections as obtained in Basic Protocol, 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 produced as outlined in Adams ( ) or purchased (e.g., ThermoFisher Scientific)
  • Fluorophore‐conjugated streptavidin
  • Additional reagents and equipment for microwave‐assisted immunocytochemistry ( protocol 9)
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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. doi: 10.1177/40.10.1527370.
  Berghorn, K.A., Bonnett, J.H., and Hoffman, G.E. 1994. cFos immunoreactivity is enhanced with biotin amplification. J. Histochem. Cytochem. 42:1635‐1642. doi: 10.1177/42.12.7983364.
  Chan, J., Aoki, C., and Pickel, V.M. 1990. Optimization of differential immunogold‐silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding. J. Neurosci. Methods 33:113‐127. doi: 10.1016/0165‐0270(90)90015‐8.
  Felten, S.Y. and Olschowka, J. 1987. Noradrenergic sympathetic innervation of the spleen: II. Tyrosine hydroxylase (TH)‐positive nerve terminals form synapticlike contacts on lymphocytes in the splenic white pulp. J. Neurosci. Res. 18:37‐48. doi:10.1002/jnr.490180108.
  Ferris, A.M., Giberson, R.T., Sanders, M.A., and Day, J.R. 2009. Advanced laboratory techniques for sample processing and immunolabeling using microwave radiation. J. Neurosci. Methods 182:157‐164. doi: 10.1016/j.jneumeth.2009.06.002.
  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. doi: 10.1016/1058‐6741(92)90021‐O.
  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. doi: 10.1177/29.4.6166661.
  Muñoz, T.E., Giberson, R.T., Demaree, R., and Day, J.R. 2004. Microwave‐assisted immunostaining: A new approach yields fast and consistent results. J. Neurosci. Methods 137:133‐139. doi: 10.1016/j.jneumeth.2004.02.020.
  Research Biochemicals International. 1998. Too much of a good thing: The importance of antibody titration. 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. doi: 10.1177/18.5.315.
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Supplementary Material