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   Home > Protein Methods Library > Overview of Immunohistochemistry


Immunohistochemistry

Introduction to Immunohistochemistry
Fixation of Tissues and Cells
Inhibition of Endogenous Tissue Components
Blocking of Nonspecific Sites
Labeled Antibodies
Avidin-Biotin Staining
Immunohistochemical Substrates
Immunohistochemical Troubleshooting
Tech Tips

Introduction to Immunohistochemistry

Immunohistochemistry (IHC) combines anatomical, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label. IHC makes it possible to visualize the distribution and localization of specific cellular components within a cell or tissue. The term immunohistochemistry is often used interchangeably with immunocytochemistry and immunostaining.

The principle of immunohistochemistry has existed since the 1930s, but it was not until 1942 that the first immunohistochemistry study was reported. Coons and his colleagues2 used FITC-labeled antibodies to localize Pneumococcal antigens in infected tissues. Since then improvements have been made in protein conjugation, tissue fixation methods, detection labels and microscopes, making immunohistochemistry a routine and essential tool in many laboratories.

Today, the most popular methods of detection are with enzyme-antibody and fluorophore-antibody conjugates(Table 11). Methods utilizing enzyme conjugated antibodies were developed independently by Nakane and Pierce3 and Avrameas and Uriel4. After the antigen-antibody reaction, the enzyme label is reacted with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. A further advantage of using enzyme labeled systems is the option to make the product electron dense for electron microscopy. Pierce offers a wide variety of reagents for the analytical development of immunohistochemical stains.

 Table 11. Typical Immunhistochemical Protocol
1. Fix tissue to be stained.
2. Block nonspecific sites with serum or blocker protein.
3. Incubate with primary antibody (1:100-1:1,000).
4. Drain reagent.
5. Incubate with secondary antibody-enzyme conjugate
(1:2,000-1:5,000).
6.Wash.
7. Incubate with substrate.
8. Visualize the stained tissue.

Fixation of Tissues and Cells

For immunohistochemical analysis, it is essential that the morphology of the tissues and cells be retained and that the antigenic sites be accessible. Tissue blocks or tissue sections are typically immersed into a fixative solution. Fixation is necessary to prevent artifactual diffusion of soluble tissue components, to arrest enzymatic activity, to avoid decomposition of the structure (Table 12) and to protect the tissue against the deleterious effects involved with the various stages of the immunohistochemical process.
Table 12. Types of Fixatives used for a Given Sample
Antigen Recommended Fixative
Most proteins, peptides and enzymes of low molecular weight Formaldehyde
Paraformaldehyde
Paraformaldehyde-picric acid
Enzymes and peptides Paraformaldehyde-picric acid
Glutaraldehyde
Small molecules such as amino acids Glutaraldehyde
Intracytoplasmic antigens; lymph node biopsies Mercuric chloride
Membrane proteins Acetic acid-zinc chloride
Glycoproteins Periodate-lysine-paraformaldehyde
Larger protein antigens (immunoglobulin) Precipitating fixatives (ethanol, methanol, acetone)

Fixatives help tissue preservation by preventing autolysis caused by lysosomal enzymes and to prevent any bacterial or fungal growth. Fixatives will often cross-link the tissue so that it can become difficult for the immunoassay reagents to penetrate the tissue. Each fixative procedure must be optimized to minimize this problem without altering the antigen or disturbing the endogenous location and the cellular detail of the tissue. The best fixative differs from tissue to tissue and from antigen to antigen. If the antigen is available in purified form, it is best to analyze the effects of different fixatives in a dot blot analysis to determine the effect of each procedure on antibody-antigen recognition8.

Several popular histochemistry and histopathology texts describe in detail the different fixatives and results on various tissue components (Table 13).5-8 To determine the appropriate fixative for a particular system, guidelines such as those in Table 13 are helpful, but each antigen is unique. While a particular fixative may preserve the immunoreactivity of one epitope, it may destroy other epitopes on the same antigen. Caution should be taken when choosing a fixative, and several aspects should be considered: type of material, rate of penetration and fixation, concentration, pH, temperature and type of post-fixation treatment.

Table 13. Formulations of Common Fixatives used in Immunohistochemistry
4% Paraformaldehyde and 0.1% Glutaradehyde in 0.1 M Phosphate Buffer
8% Paraformaldehyde stock solution, 499 ml
0.2 M Phosphate buffer, pH 7.4, 499 ml
Mix together.
Add 50% glutaraldehyde, 2 ml
Use solution immediately.
5% Glutaraldehyde in 0.1 M Cacodylate and 1% Sodium Metabisulfite, pH 7.5-7.8
Sodium cacodylate, 16.0 g
Sodium metabisulfite, 10.0 g
Distilled water, 800 ml
Stir until dissolved.
Add 50% glutaraldehyde, 100 ml
Stir until mixed.
pH to 7.5-7.8
Bring volume up to a final 1 liter with distilled water.
Use solution immediately.
Bouin’s Fixative
Filtered, saturated aqueous picric acid, 750 ml
40% Formalin, 250 ml
Glacial acetic acid, 50 ml
Stir until mixed.
Store at room temperature.
10% Phosphate Buffered Neutral Formalin
40% Formalin, 100 ml
Dibasic sodium phosphate, anhydrous, 6.5 g
Monobasic sodium phosphate, monohydrate, 4.0 g
Distilled water, 900 ml
Stir until dissolved.
Store at 4°C.
Mercuric chloride-based fixatives include Zenker’s Solution and B5.
Zenker’s Stock Solution
Mercuric chloride, 50 g
Potassium dichromate, 25 g
Sodium sulfate, 10 g to 1 liter with distilled water
Zenker’s Working Solution
Zenker’s Stock Solution, 100 ml
Glacial acetic acid, 5 ml
B5 Stock Solution
Mercuric chloride, 12 g
Sodium acetate, anhydrous, 2.5 g
Distilled water, 200 ml
B5 Working Solution
B5 Stock Solution, 20 ml
40% Formalin, 2 ml
Use immediately.

Formaldehyde-based fixatives stabilize tissues by reacting primarily with basic amino acids to form methylene cross-links. The number of methylene bridges formed depends on the concentration of formaldehyde, temperature, pH and time of exposure. Formaldehyde fixatives are relatively mild and are partially reversible. They are well tolerated by tissues and allow for good penetration. The disadvantages of using this type of fixative include shrinkage and distortion of the tissue and, therefore, the antigenic sites. Examples of formaldehyde-based fixatives include Bouin’s solution and 10% phosphate buffered neutral formalin.

Glutaraldehyde-based fixatives were first introduced by Sabatini1. Glutaraldehyde is a dialdehyde that reacts with amino groups, sulfhydryl groups and possibly with aromatic ring structures. Fixatives containing glutaraldehyde are stronger cross-linkers of protein than formaldehyde. They penetrate tissue more slowly, causing extraction of soluble antigens and modification of the structure. Tissues that have been fixed with a glutaraldehyde-based fixative must be treated before the immunoassay with inert amine-containing molecules because free, unsaturated aldehyde groups are available to covalently link amine-containing moieties such as antibodies. The most efficient aldehyde blockers are ethanolamine and lysine2. Glutaraldehyde-based fixatives include 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer and 5% glutaraldehyde in 0.1 M cacodylate and 1% sodium metabisulfite, pH 7.5-7.8.

An alternative to glutaraldehyde-based fixatives is a diimidoester fixative using Dimethyl Suberimidate (DMS) (Product # 20700).9 DMS is a bifunctional reagent that cross-links by reacting with the a- and e-amino groups of proteins. Diimidoesters are unique in that they carry an amido group next to the functional groups on the molecules. As a result, DMS does not affect the net charge of the protein. The advantages of using DMS as a fixative for both light and electron microscope studies include retention of immunoreactivity of the antigen and the lack of aldehyde groups that require blocking.

Mercuric chloride-based fixatives are used as an alternative to formaldehyde-based fixatives to overcome poor cytological preservation. These fixatives work by additive and coagulative properties. The major advantages include: good penetration, resulting in more intense immunostaining; and the preservation of cytological detail, allowing for easier morphological interpretation. They often contain neutral salt to maintain tonicity and can be mixed with other fixatives to provide a balanced solution. Mercuric chloride-based fixatives include Zenker’s Solution and B5. Sections must be cleared of mercury deposits before immunostaining.

Precipitating fixatives include ethanol, methanol and acetone. They precipitate large protein molecules and are good for cytological preservation. They result in poor penetration and possibly incomplete fixation. They are not good for electron microscope work because they cause tissue shrinkage.
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Inhibition of Endogenous Tissue Components

Tissues, being complex biological systems, can contain materials that react with the various reagents used to develop an immunohistochemical stain. Cellular components such as enzymes and the vitamin, biotin, can result in unwanted signals, falsely indicating the presence of an antigen. The immunoperoxidase method of signal detection is commonly used for the immunostaining of cells and tissues. Final analysis of this staining method is complicated by the presence of endogenous peroxidase and “pseudoperoxidase” activity in the cells and tissues. Peroxidase reacts with hydrogen peroxide to reduce the diaminobenzidine (DAB) substrate or other peroxidase substrates, resulting in nonspecific staining of the tissue or false positives. To determine if there is endogenous peroxidase activity, the fixed tissue should be reacted with a peroxidase substrate such as DAB (Product # 34001, 34002). Any colored precipitate that forms in the tissue from this treatment indicates endogenous activity. If a tissue is rich in peroxidase activity, an alternative enzyme label, such as calf intestinal alkaline phosphatase (AP), can be used.

Several methods have been devised to inhibit or destroy endogenous peroxidase activity after tissue fixation. The most common methods are 3% H2O210 or H2O2 in methanol.11 Pierce offers a peroxidase inhibitor, ImmunoPure Peroxidase Suppressor (Product # 35000), which more effectively inhibits peroxidase activity than H2O2 or H2O2 in methanol (Figure 15). Activity of an antibody is not affected by the addition of this inhibitor (Figure 16). To ensure that the peroxidase inhibitor does not affect immunoreactivity of the primary antibody, apply the inhibitor after the incubation with the primary antibody and before incubation with the peroxidase conjugate.12
   
Figure 15. Suppression of Horseradish  Peroxidase.  Figure 16. Immunopure Peroxidase Inhibitor Effect on Antibody Binding. 

Avidin-biotin staining methods are commonly performed to enhance the detection of a target antigen. Because free or labeled avidin is used in these systems, a problem arises in tissues that are rich in endogenous biotin including liver, mammary gland, adipose tissue and kidney.13 Endogenous avidin activity is most pronounced in cryostat sections. To suppress this endogenous avidin activity it is necessary to block the endogenous biotin with avidin (Product # 21121) and then block the avidin sites with biotin.14 Since the avidin molecule contains 10% carbohydrates, binding to lectin-like substances in tissues is possible. To block this binding, the addition of an analog to the carbohydrate on avidin can be used.15 Alternatively, replacing avidin with streptavidin (Product # 21122, 21125) or NeutrAvidin (Product # 31000), which does not contain carbohydrates, will eliminate this interaction.

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Blocking of Nonspecific Sites

Blocking the reactive sites in tissues is essential for the development of an immunohistochemical reaction. Normal serum is the most popular blocking agent for immunohistochemical staining. The principle is that non-immune serum from the host species of the secondary antibody is applied to the tissue at the beginning of the procedure and will adhere to protein-binding sites either by nonspecific adsorption or by binding of specific, but unwanted, serum antibodies to antigens in the tissue. If a direct technique is being performed, the blocking serum should be from the host species providing the primary antibody. Pierce offers a variety of normal sera including goat (Product # 31872, 31873), horse (Product # 31874), human (Product # 31876), mouse (Product # 31880, 31881), rabbit (Product # 31883, 31884), rat (Product # 31888) and swine (Product # 31890).

When blocking the unreactive sites on fixed tissue, the blocking buffer should be incubated for 10-30 minutes prior to adding the primary antibody. To prevent removal of the blocking buffer, do not wash the tissue, merely drain off the blocking buffer. Logically, it should be reapplied after washing off the primary antibody and before adding the secondary antibody conjugate to prevent nonspecific binding of the secondary antibody. This is not necessary if the blocking buffer is added to the secondary antibody as a carrier protein. Adding a detergent such as 0.05% Tween-20 (Product # 28320) with the carrier protein (serum protein) may help to reduce background staining by reducing any hydrophobic interactions between tissue and reagent proteins.

Labeled Antibodies

Pierce offers a wide variety of ImmunoPure labeled polyclonal antibodies for use in immunohistochemistry. The labels include biotin, fluorescein, rhodamine, horseradish peroxidase and calf intestinal alkaline phosphatase. For a complete selection of labeled polyclonal antibodies please refer to the Antibody Selection Guide. Pierce offers affinity purified antibodies directed against immunoglobulins from many different species and adsorbed against other species to minimize cross-reactivity in single- (direct method) and double-labeling (indirect method) protocols.
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Avidin-Biotin Staining

Staining intensity is a function of the enzyme activity and improved sensitivity can be achieved by increasing the number of enzyme molecules bound to the tissue. The multiple binding sites between the tetravalent avidin and biotinylated antibodies (bound to the antigen) are ideal for achieving this amplification. The avidin-biotin complex (ABC) method was developed by Hsu and colleagues.16-18 Figure 17. A biotinylated enzyme (HRP or AP) is pre-incubated with avidin, forming large complexes to be incubated with the biotinylated antibody. Typically, the avidin and biotinylated enzyme are mixed together in a specified ratio for about 15 minutes at room temperature to form the complex. An aliquot of this solution is then added to the tissue, and any remaining biotin-binding sites on the avidin bind to the biotinylated antibody that is already bound to the tissue. The result is a greater concentration of enzyme (3 enzyme molecules to one avidin molecule) at the antigenic site and therefore an increase in signal intensity and sensitivity (Table 14).
Table 14. Important Features of the ABC Method for Immunohistochemical Staining

Advantages of the ABC Method

Disadvantages of the ABC Method
  • Increased enzyme label at the tissue antigen site
  • Increased detection efficiency
  • Requires less primary antibody
  • Reduced assay time compared to the PAP method
  • Some tissues may require blocking of
    endogenous biotin to avoid nonspecific staining
  • The ABC complex is large, hindering tissue
    penetration in some applications

Pierce offers a line of avidin-biotin immunostaining kits that have optimized the ABC method. ImmunoPure ABC Staining Kits are available with both HRP and AP detection systems. The proper kit is chosen based on the species of primary antibody used. For example, if the primary antibody is an IgG produced in mice, the kit selected should be an ImmunoPure ABC kit designated “mouse IgG.”



Figure 17. ABC method.

 


Figure 18. LAB method.

Each kit includes 3 ml of the appropriate blocking serum, 1 ml of the biotinylated affinity purified secondary antibody and 2 ml each of the avidin and biotinylated enzyme reagents. The kits require only the addition of a specific primary antibody and an enzyme substrate. Pierce offers a full line of substrates for the development of the tissue stain. The ABC method is very popular because of the high sensitivity and low background staining achieved with its use.

If the avidin-biotin complex becomes too large to penetrate the tissue, a second method developed by the Guesdon and colleagues19 can be used. The labeled avidin-biotin (LAB) method Figure 18 employs an avidin-enzyme conjugate (or streptavidin-enzyme conjugate) to detect the bound biotinylated-primary antibody on the tissue section. This smaller complex allows better tissue penetration. Pierce offers carbohydrate-free, NeutrAvidin and streptavidin enzyme conjugates as superior alternatives to avidin for reduced background and improved sensitivity.

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Immunohistochemical Substrates

Immunoenzymatic staining of tissues results from the reaction of a soluble substrate with an enzyme to produce an insoluble, colored product. The intensity of the color produced when the substrate is added should correlate to the concentration of the primary antibody and the respective tissue antigen. Many enzymes are used for these applications, but the most common selections are horseradish peroxidase and calf intestinal alkaline phosphatase. Enzymatic activity is dependent on several variables: concentration of enzyme and substrate, buffer, pH, temperature, and possibly light.

Pierce offers a variety of substrates in both a powdered form and in substrate kit form for immunohistochemical studies (Table 15). The most sensitive HRP substrate Pierce offers is the Metal Enhanced DAB substrate (Table 16), which is 50 times more sensitive than a general DAB (diaminobenzamidine) substrate.20 This formulation produces a stronger signal than DAB alone due to the deposition of cobalt and nickel which forms a dark brown/black precipitate (see Figure 19).

Table 15. Substrates for Immunohistochemistry
Substrate Format Enzyme Reporter Features Sensitivity Signal Color Product #
Metal Enhanced DAB 2-Component Reagent Kit HRP-Peroxidase 50 times more sensitive than DAB without enhancement Highest Brown-black precipitate 34065
1-Step TMB Single Step, Ready-to-Use Reagent HRP-Peroxidase Low background for high sensitivity High Dark blue precipitate 34018
1-Step 4-CN Single Step, Ready-to-Use Reagent HRP-Peroxidase Low background for high sensitivity High Blue-purple precipitate 34012
CN/DAB 2-Component Reagent Kit HRP-Peroxidase Low background for high sensitivity High Black precipitate 34000
DAB Dry Powder HRP-Peroxidase Can be formulated Medium Brown precipitate 34001
1-Step NBT/BCIP Single Step, Ready-to-Use Reagent Alkaline Phosphatase Low background for high sensitivity High Black-purple precipitate 34042
1-Step NBT/BCIP + Suppressor Single Step, Ready-to-Use Reagent Alkaline Phosphatase Contains levamisole for inhibition of endogenous peroxidases High Black-Purple precipitate 34070
BCIP Dry Powder Alkaline Phosphatase Can be formulated Medium Blue-purple precipitate 34040
NBT Dry Powder Glucose Oxidase & Alkaline Phosphatase Non-carcinogenic Medium Blue-purple precipitate 34035
X-Gal Dry Powder ß-Galactosidase Can be used in conjunction with IPTG to detect vectors, plasmids or DNA fragments that express the ß-Gal gene High Bright blue indigo precipitate 34050
IPTG Dry Powder ß-Galactosidase Can be used in conjunction with X-Gal to detect vectors, plasmids or DNA fragments that express the ß-Gal gene High Blue-purple precipitate 34060


 

 

Prostatic Acid Phosphatase stained with Pierce Metal Enhanced DAB and blocked with SuperBlock Blocking Buffer in TBS
 

Prostatic Acid Phosphatase stained by original DAB method (Graham and Karnovsky, 1966) 

Figure 19. Comparison of staining intensity between Metal Enhanced DAB (Product # 34065) and DAB Substrates.

Table 16. Typical Immunohistochemical Protocol Using Metal Enhanced DAB Substrate
  1. Prepare and fix tissue.
  2. Block nonspecific sites in the tissues with normal serum or other blocking buffer such as SuperBlock Blocking Buffer in TBS (Product # 37535) for 30 minutes at room temperature in a humidity chamber. Pour off the blocking buffer but do not remove excess.
  3. Incubate the tissues with the primary antibody for 30 minutes at room temperature in a humidity chamber.
  4. Wash the tissues in buffer for 3 minutes. Repeat wash step. Remove excess wash buffer.
  5. Suppress endogenous peroxidase activity using Pierce ImmunoPure Peroxidase Suppressor (Product # 35000).
  6. Incubate for 15-30 minutes at room temperature in a humidity chamber.
  7. Wash the tissues in buffer for 3 minutes. Repeat wash step. Remove excess wash buffer.
  8. Incubate the tissues with HRP-conjugated secondary antibody for 30 minutes at room temperature in a humidity chamber or with the ABC System (Product # 32020, 32050).
  9. Wash the tissues in buffer for 3 minutes. Repeat wash step. Remove the excess wash buffer.
  10. Add the Metal Enhanced DAB Substrate and incubate until desired staining is achieved. (Typical incubations range from 5 to 15 minutes.)
  11. Stop development by washing tissue with ultrapure water.


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Immunohistochemical Troubleshooting

In immunohistochemical techniques, there are several steps prior to the final staining of the tissue antigen, and many potential problems affect the outcome of the procedure. The two major areas that must to be addressed are a poor signal:noise ratio and poor staining. Patience is required to solve these problems.

Poor Signal:Noise Ratios
To improve poor signal:noise ratios, the following procedures are recommended:
  • Optimize fixation. Each tissue antigen will react differently with different fixatives. Optimize the pH, incubation time and temperature.
  • Take great care in preparing and processing the tissue, because damage to the tissue can cause diffuse staining.
  • Inhibit endogenous activity. Many tissues are high in endogenous peroxidase, especially cryostat sections. Using an inhibitor such as the Pierce ImmunoPure Peroxidase Suppressor (Product # 35000) before adding the HRP eliminates endogenous peroxidase activity. Adding levamisole to AP substrates will inhibit endogenous AP. When using an avidin-biotin system, caution should be taken to block all endogenous avidin binding activity.
  • Optimize the blocking agent used for the tissue section. Using a nonimmune serum from the host of the labeled antibody or a blocking buffer is essential to block the nonspecific binding sites in the tissues.
  • After the tissue has been incubated with the blocking buffer, do not rinse off the blocking agent; simply drain the excess.
  • Use a monoclonal primary antibody instead of a polyclonal to reduce cross-reactivity.
  • Decrease the amount of label used (dilute the labeled primary antibody or secondary antibody).
  • Use cross-absorbed polyclonal antibodies to reduce cross-reactivity.
  • Affinity purify the antibody preparation on an immobilized antigen column.
  • If there is a hydrophobic interaction between the tissue proteins and antibody molecule, decrease the ionic strength of the diluent used for the antibody, include a detergent or add the blocking protein to the diluent.
  • If there is an ionic interaction between the tissue proteins and the antibody molecule, increase the ionic strength of the diluent.
  • Decrease the incubation times with the primary and secondary antibodies to reduce nonspecific binding.
  • If a fluorescent marker is being used, check to make sure that there is no autofluorescence in the unprocessed, fixed tissue. If there is autofluorescence, choose a fluorescent marker that will not compete.
  • Choose a substrate that will produce a higher signal:noise ratio for the system such as Metal Enhanced DAB rather than DAB.
  • If the tissue is not being penetrated well, try preparing thinner sections or using unmasking agents.
Poor Signal
To increase signal, the following procedures are recommended:
  • Optimize fixation. The immunoreactivity can be affected by the fixative step along with the processing step. Avoid freeze/thaw cycles and high temperatures if the antigen is susceptible.
  • Use clean slides for mounting of tissue, and use appropriate conditions to prevent tissue from being removed during processing.
  • If an AP system is being used, do not use phosphate buffer. If an HRP system is being used, do not use sodium azide. Both will inhibit the enzyme activity.
  • Do not over-block the tissue, since antigenic sites may be masked.
  • Screen monoclonal antibodies using a membrane system such as the Easy-Titer ELIFA Unit (Product # 77000) instead of the typical ELISA method. Polystyrene plates used to screen monoclonal antibodies alter the conformation of proteins bound to the surface. It is possible that monoclonal antibodies selected in an ELISA will not recognize native protein in the tissue.
  • Increase penetration of the tissue by using unmasking agents such as trypsin, pepsin, chymotrypsin, and Pronase.
  • Increase the detection efficiency, and possibly the sensitivity, by using signal amplification systems such as ABC.
  • Increase incubation times or concentrations of the primary or secondary reagents.
  • Use a more sensitive substrate system such as the Metal Enhanced DAB Substrate.
  • Always run a positive control to determine if the system is working.
The above are suggestions only. Controls should always be used to determine where there may be a problem. Optimization for each immunohistochemical system is essential. The time-consuming optimization steps will alleviate frustration over poor signal:noise ratio and poor staining.

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References
  1. Marrack, J. (1934). Nature 133, 292-293.
  2. Coons, A.A., et al. (1942). J. Immunol. 45, 159-170.
  3. Nakane, P.K. and Pierce, G.B. (1966). J. Histochem. Cytochem. 92, 452-455.
  4. Avrameas, S. and Uriel, J. (1966). C.R. hebd. Seanc. Acad. Sci. (D) Paris 262, 2543-2545.
  5. Culling, C.F.A. (1974). Handbook of Histopathological and Histochemical Techniques, 3rd ed. Butterworths, London.
  6. Lillie, R.D. and Fullmer, M. (1976). Histopathologic Technique and Practical Histochemistry, 4th ed. McGraw Hill Book Co., New York.
  7. Pearse, A.G.E. (1980). Histochemistry, Theoretical and Applied, 4th ed., Vol 1. Preparative and Optical Technology. Churchill Livingstone, London.
  8. Larsson, L.I. (1988). Immunocytochemistry: Theory and Practice. CRC Press, Boca Raton, FL.
  9. Hassel, J. and Hand, A.R. (1973). J. Histochem. Cytochem. 22, 229-239.
  10. Köller, U., et al. (1986). J. Immunol. Methods 86, 75.
  11. Streefkerk, J.G. (1972). Nature 330, 80.
  12. Fink, B., et al. (1979). J. Histochem. Cytochem. 27, 1299.
  13. Dakshinamurti, K. and Mistry, S.P. (1963). J. Biol. Chem. 238, 294.
  14. Wood, G.S. and Warnke, R. (1981). J. Histochem. Cytochem. 29, 1196-1204.
  15. Naritoku, W.Y. and Taylor, C.R. (1982). J. Histochem. Cytochem. 30, 253-260.
  16. Hsu, S.M., et al. (1981). Am. J. Clin. Path. 75, 816.
  17. Hsu, S.M., et al. (1981). J. Histochem. Cytochem. 29, 577-580.
  18. Hsu, S.M., et al. (1981). Am. J. Clin. Path. 75, 734-738.
  19. Guesdon, J.L., et al. (1983). J. Histochem. Cytochem. 27, 1131.
  20. Adams J.C., (1981). J. Histochem. Cytochem. 29, 775.

Tech Tips
Protein stability and storage
Block endogenous biotin

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