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Immunohistochemistry (IHC) Guide and Troubleshooting


- Immunohistochemistry
- Selecting Antibodies
- Sample preparation - fixation
- Sample preparation - embedding
- Antigen Retrieval
- Immunostaining
- No staining
- High background
- Non-specific staining
Related Information:
- Comparison of formaldehyde and formalin


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Immunohistochemistry is a laboratory technique utilized for the visual detection of antigens in tissue. When working with cells this technique is generally referred to as immunocytochemistry. These techniques are widely used by histology/pathology laboratories and life science researchers as a method to detect abnormal cells or to characterize the localization of proteins within a biological sample. Visual detection is achieved using antibodies that have been chemically conjugated with a marker such as a fluorescent dye, enzyme, radioactive element or colloidal gold. The final antibody-antigen reaction is visible using a light or fluorescent microscope depending on the labeled antibodies used for detection.
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Selecting Antibodies:

There are numerous factors to consider when choosing a primary antibody as there are advantages and disadvantages of using a monoclonal or polyclonal primary antibody. Monoclonal antibodies are generated by a single B-cell clone from one animal which results in a homogenous population of immunoglobulin directed against a single epitope. Polyclonal antibodies are a developed by multiple B-cell clones of the animal, which results in a heterogeneous mixture of antibodies directed against various epitopes of the same antigen.

Generally researchers may choose a monoclonal antibody because batches produced from the established hybridoma line allow for identical production, thus standardization. Additionally, they are less likely to have background staining and they are less likely to have cross-reactivity with other proteins. Since polyclonal antibodies recognize multiple epitopes on the same antigen, they are more tolerant of minor changes in the antigen during fixation and processing. Furthermore, polyclonal antibodies can boost the detection signal of the antigen and are the preferred method when using denatured proteins.

The species the antibodies were raised against also needs to be considered when choosing antibodies for IHC. To provide the lowest backgrounds, the primary and the secondary antibodies should be raised against the same species.
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Sample Preparation: Fixation

The first step is collecting the tissue sample. Properly collecting the tissue is an extremely important step because it will avoid sample degradation and maintain quality. Factors that may affect the quality include time until fixation and the temperature of the sample. Additionally, the tissue should be placed in a fixative solution as soon as possible.

Fixatives are used to immobilize antigens while retaining cellular and subcellular structures through cross-linking methylene bridges and Schiff bases between basic amino acid residues of proteins. There are a variety of fixatives used for IHC and there isn’t one that is ideal for all antibodies and applications. The most common fixative used is 10% neutral buffered formalin. Others include methanol, ethanol and acetone which may be used for fixation and permeabilizing the membrane.
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Sample Preparation: Embedding

There are also various embedding compounds that can be used to support the tissue during processing/sectioning. Frozen sections are ideal for quick processing and situations where the antibody may not be compatible with paraffin compounds. Frozen sections are typically used for direct or indirect immunofluorescence detection methods and sectioned at a thickness of 10 microns. The second type and most common of embedding media is paraffin. Paraffin is used for maximum tissue preservation and makes a stronger block for sectioning tissue. Typically paraffin sections are cut between 3-5 microns thick.
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Antigen Retrieval:

Unmasking the antigen is sometimes necessary because the process of paraffinization and using strong fixatives may inhibit the primary antibodies attachment to the antigen. There are two common processes for antigen retrieval: heat-mediated and enzymatic digestion. Both of these techniques serve to break the cross-linkages and expose the antigen. Additionally, a deparaffinization step is used on paraffin embedded tissue sections through a process of series of alcohol washes.

When using the enzymatic digestion reagents it is important to not over or under digest – the digestion time should be optimized. Enzymes used include pronase, trypsin, pepsin and proteinase K. Heat induced antigen retrieval can be done using a pressure cooker, microwave, autoclave or water bath.
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Immunostaining is the process of incubating the tissue/cells with antibodies to form the antigen-antibody complex for the detection/signal of targeted antigen. Detection is commonly achieved using a colorimetric (enzyme) reaction or a fluorescent signal generated from an ultraviolet source.

There are multiple labeling techniques including direct, indirect and indirect with signal amplification. Direct labeling uses a primary antibody directly conjugated to a signaling source, while indirect labeling has a signal generating secondary antibody that will attach to the primary antibody making contact with the antigen. For example, a primary antibody produced from rabbit would be detected using a secondary antibody that is anti-rabbit and conjugated to a detecting probe. Finally, the indirect with signal amplification technique is accomplished using a biotinylated secondary antibody and an amplification reagent such as streptavidin.

Immunofluorescence techniques use antibodies chemically conjugated to fluorescent dyes that are visible under a UV light. Two popular dyes are Fluorescein Isothiocyanate (FITC) and Rhodamine Isothiocyanate (TRITC).

Colorimetric reactions use antibodies chemically conjugated to the enzymes horseradish peroxidase or alkaline phosphatase. The enzyme catalyzes the reaction of the substrate forming a color precipitate. Common substrates include Diaminobenzidine (DAB), 3-Amino-9-Ethylcarbazole (AEC), 4-Chloro-1-Naphthol (4-CN) and Bromochloroindolyphosphate/Nitro Blue Tetrazolium (BCIP/NBT).
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Common problems encountered include insufficient staining, too much staining or high background.

 No staining

Problem Possible Solution(s)
The primary antibody and the secondary antibody may not match. It is recommended that the secondary antibody be against the species in which the primary antibody was raised (e.g if the primary antibody is raised in mouse, use anti-mouse secondary antibody).
The antibody may not be suitable for IHC procedures which reveal the protein in its native (non denatured) form. Test the antibody on a non-denatured and denatured western blot to make sure the antibody is recognizing non-denatured antigen.
The secondary antibody was not stored in the dark. It is recommended to avoid exposing the secondary antibody to light.
Not enough primary antibody is binding the protein of interest. Dilute the antibody less.
incubate for longer at 4°C.
The protein of interest is not abundantly present in the tissue. Run a positive control.
Utilizing an amplification step can assist in amplifying the signal.
Use a lower dilution of primary antibody.
The protein of interest is a nuclear protein and the antibody cannot penetrate the nucleus. To facilitate penetration of the nucleus, add a permeabilizing agent to the blocking buffer and the antibody dilution buffer.
Deparafinization may be insufficient Deparaffinize sections longer.
Use freshly prepared xylene.
Fixatives such as formalin and paraformaldehyde may be modifying the epitope the antibody recognizes. Use antigen retrieval method to unmask the epitope.
Fix for less time.
The PBS buffer may be contaminated with bacteria that has damaged the protein of interest. Use a preservative such as 0.01% azide in the PBS antibody storage buffer.
use fresh sterile PBS.

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High background

Problem Possible Solution(s)
Blocking of non-specific binding may be insufficient. Increase the blocking incubation period to 30 min for sections and 1 hour for cell culture. Use a blocking agent such 10% normal serum for sections and 1-5% BSA for cell culture.
The primary antibody concentration may be too high. Titrate the antibody to a more optimal concentration, incubate for longer using more dilute. This will provide a slower but more specific binding.
The secondary antibody may be binding non-specifically. Run a secondary control without primary antibody.
Incubation temperature may be too high. Incubate tissue sections or cells at 4°C.
Tissue not adequately washed. Wash extensively with PBS between all steps
Endogenous peroxidases are active. Use enzyme inhibitors such as Levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.
Fixatives such as formalin and paraformaldehyde are too strong and may have modified the epitope the antibody recognizes. Change antigen retrieval methods.
Decrease the incubation time with the antigen unmasking solution.
Too much substrate was applied. Reduce substrate incubation time.
The chromogen reacts with the PBS present in the cells. Use a Tris buffer to wash sections prior to incubating with the substrate.
Pemeabilization has damaged the membrane and removed the membrane protein. Remove permeabilizing agent from your buffers.

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Non-specific staining

Problem Possible Solution(s)
The concentration of the primary or secondary antibody may be too high.
Try decreasing the antibody concentration and/or the incubation period. Compare signal intensity against cells that do not express the target.
The primary antibody is raised against the same species as the tissue stained (e.g rat primary antibody tested on rat tissue). When the secondary antibody is applied, it binds to all the tissue since it was also raised against that species.
Use primary/secondary antibodies raised against a different species than your tissue.
Endogenous peroxidases are active. Use enzyme inhibitors such as Levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.
The sections/cells have dried out. Keep tissue sections and cells at high humidity and do not let them dry out.

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Related Information:

Comparison of Formaldehyde and Formalin:

Formaldehyde is a colorless gas known by the chemical formula of CH2O.

When formaldehyde gas is dissolved in water it saturates into solution at 37% (by weight) or 40% (by volume) and is referred to as 37-40% formaldehyde, 100% formalin or strong formalin.  The word formalin is used to refer to solutions of formaldehyde gas dissolved in water.  Stored formalin contains approximately 10% methanol to slow down polymerization that leads to precipitation of paraformaldehyde.  Commonly used solutions of formalin as a fixative include:
   •   10% NBF (neutral buffered formalin):
         - 100 mL 100% formalin
         - 900 mL water.
         - 4 grams sodium dihydrogen phosphate, monohydrate (NaH2PO4H2O)
         - 6.5 grams disodium hydrogen phosphate, anhydrous (Na2HPO4)
         - The final concentration contains 4% formaldehyde.
   •   Formalin Saline:
         - 100 mL 100% formalin
         - 900 mL water
         - 8.5 grams sodium chloride.
   •   10% Formalin:
         - 100 mL 100% formalin
         - 900 mL water

Paraformaldehyde is the polymerized form of formaldehyde.  It’s used to make methanol-free solutions of formalin that don’t contain formic acid when first made.
   •   4% Paraformaldehyde:
         - 40 grams of paraformaldehyde
         - Make to one liter with phosphate buffered saline (PBS)
         - Heat solution under fume hood to 60ºC - 70ºC and add drops of 10M NaOH until solution  clears.  Filter final solution.

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