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

  •   FigureFigure 2.12.1 Immunocytochemical method that employs a directly tagged secondary antibody (pink). As illustrated, the secondary antibody is labeled with a fluorescent molecule, but this same approach can be used with enzymes attached.
  •   FigureFigure 2.12.2 The avidin‐biotin complex (ABC) method for immunofluorescence. Note that, compared to the use of a direct fluorophore‐linked secondary (Fig. ), this method enables more molecules of fluorophore to be attached to the complex.
  •   FigureFigure 2.12.3 The ABC peroxidase technique. This approach is similar to that shown in Figure , but substitutes a biotinylated peroxidase. When incubated with a substrate such as DAB (lower left) or NiDAB (lower right), the colored insoluble product is deposited to sites near the enzyme. The micrographs (insets) show an example of each of the chromogens (staining for luteinizing hormone releasing hormone/gonadotrophin releasing hormone).
  •   FigureFigure 2.12.4 TSA‐amplified fluorescence using biotinylated tyramine and streptavidin fluorophore. Note the greatly increased number of fluorescent molecules compared with that seen for either fluorophore‐tagged secondaries or ABC streptavidin methods. P = peroxidase.
  •   FigureFigure 2.12.5 Variation in the color of the NiDAB product when high versus low concentrations of the primary antibody are used. Low‐power micrographs show melanin‐concentrating hormone (MCH) in the lateral hypothalamic area (LHA) using the anti‐MCH at 1:3,000 and 1:150,000. Note that at the high concentration of the antibody (A), the staining is uneven and in some regions has very high background compared with that seen when the primary antibody concentration is reduced (B). The series along the bottom (C to F) provides higher magnification and a more complete titration series that illustrates how the signal‐to‐noise ratio increases and the color shifts from brown to blue‐black as the antibody is diluted to its optimum. Scale bar for A and B = 250 µm; C‐F = 50 µm. Abbreviations: ic = internal capsule, f = fornix, opt = optic tract.
  •   FigureFigure 2.12.6 Titrations of anti‐MCH (melanin‐concentrating hormone) using different immunofluorescence methods. Fluorescence photomicrographs show MCH‐immunoreactive cells in the lateral hypothalamic area using Cy‐2 conjugated directly to the secondary antibody (AE), a biotinylated secondary antibody plus streptavidin‐Cy‐2 (FI), and TSA amplification of biotinylated tyramine followed by the same Cy‐2‐conjugated streptavidin (JL). The values above the pictures refer to the absolute concentrations of the primary antibody used (left) and the concentrations of the primary antibody relative to the optimal concentration determined with ABC immunoperoxidase and NiDAB as shown in Figure . The values on the right indicate the exposure times in milliseconds for acquiring the images in the series. Some increased detection could have been obtained if longer times were used, but times in a series were held constant to stress signal‐intensity changes. All labeling was conducted from tissue obtained from the same animal. Scale bar = 50 µm.
  •   FigureFigure 2.12.7 The procedures used to perfuse a rat. The rat is anesthetized and an inverted “T”‐shaped incision made (A). The diaphragm is cut and the rib cage opened to expose the heart (B and C). After injecting heparin into the heart at its apex, a catheter is inserted into the heart as shown in C. The tip of the catheter is eased into the proximal portion of the aorta as shown in D. The right atrium is cut (as illustrated in B to enable the perfusate to drain from the animal. (Photograph courtesy of Emmanuel Díaz, a student at the Ricardo Miledi Neuroscience Course, Universidad Nacional Autónoma de México, 2008).
  •   FigureFigure 2.12.8 Example of titrations of two purchased antisera (both rabbit polyclonal antisera) against green fluorescent protein (GFP). Both are tested in the same tissue. Note that the antiserum used in the series on the left (purchased from Novus) shows specific staining for GFP in neurons of the cortex, whereas only background labeling is found in the series on the right that used the other antiserum.
  •   FigureFigure 2.12.9 Comparisons of staining for serotonin in horizontal sections in brainstem tissue that was fixed with buffered 4% paraformaldehyde with added 2.5% acrolein, versus that fixed with only buffered 4% paraformaldehyde. The inclusion of acrolein in the fixative impairs recognition of the tissue serotonin by the antibody.

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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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