IHC / ICC / IF - General Tips/Procedures and Troubleshooting

Immunohistochemistry (IHC)

Immunohistochemistry is a method for demonstrating the presence and location of proteins in tissue sections. Though less sensitive quantitatively than immunoassays such as Western blotting or ELISA, it enables the observation of processes in the context of intact tissue. This is especially useful for assessing the progression and treatment of diseases such as cancer. Immunohistochemical staining is accomplished with antibodies that recognize the target protein. Since antibodies are highly specific, the antibody will bind only to the protein of interest in the tissue section. The antibody-antigen interaction is then visualized using either chromogenic detection, in which an enzyme conjugated to the antibody cleaves a substrate to produce a colored precipitate at the location of the protein, or fluorescent detection, in which a fluorophore is conjugated to the antibody and can be visualized using fluorescence microscopy. Several type of sections are used to perform IHCs.

Frozen Sections (FS)
There are two types of cryostat sections, which include (1); fresh or unfixed sections, where quickly frozen (snap frozen) tissues are first cut, then either air-dried or fixed prior to staining; and (2) fixed frozen sections, where the tissue is first fixed then cryoprotected with sucrose or other stabilizers (to stabilize the tissue cell structure) prior to freezing and sectioning. The advantages of frozen sections are that they allow excellent antigen preservation, they are typically faster to perform, and they offer flexibility, since any fixative can be used, thereby facilitating the optimization of fixative for each antigen. However frozen sections give less morphological detail and resolution than other methods.

Paraffin Sections (PS)
The largest proportion of samples used in immunostaining are embedded in paraffin because it provides for excellent morphological detail and resolution. Modern "paraffin" is typically a mixture of paraffin wax and resin. It is an excellent embedding medium because it can be heated to liquid state, and dissolved by xylene for infiltrating the tissue, and then relatively quickly turned to a solid state again for maximum structural support during sectioning. Once mounted, the slides can be stored indefinitely until immunostaining is required; then the paraffin must be removed from the tissue to allow the water-based buffers and antibodies to penetrate.

Resin Sections (RS)
Embedding tissues in resin offers several advantages over frozen or paraffin sections, thinner sections may be used with improved morphology and less tissue shrinkage. 


Immunocytochemistry (ICC)

Immunocytochemistry differs from immunohistochemistry in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. This includes cells grown within a culture, deposited from suspension, or taken from a smear. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue.


Immunofluorescence (IF)

Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample. Immunofluorescence is a widely used technique of immunostaining and is a specific technique of immunohistochemistry and immunocytochemistry that makes use of fluorophores to visualise the location of the antibodies.


Immunohistochemistry (Paraffin Sections) – General Tips/Procedures

IHC (PS) refers to the staining of tissues that have been fixed (usually in neutral buffered formalin) and then embedded in paraffin before being sectioned. The basic steps are as follows:

1. Fixing and embedding the tissue
2. Cutting and mounting the section
3. Deparaffinizing and rehydrating the section
4. Antigen retrieval
5. Immunohistochemical staining
6. Dehydrating and stabilizing with mounting medium
7. Viewing the staining under the microscope

General Tips

1. Fixation
Proper fixation is key for the success of immunohistochemistry.10% neutral buffered formalin (NBF) is most commonly used. The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18-24 hours seems to be ideal for most applications. Under-fixation can lead to edge staining, with strong signal on the edges of the section and no signal in the middle; over-fixation can mask the epitope.

2. Cutting and Mounting
After fixation, the tissue block is embedded in paraffin, then cut in a microtome to the desired thickness (approximately 5 microns is ideal for IHC) and affixed onto the slide. Tissue sections are best mounted on positively charged or APES (amino-propyl-tri-ethoxy-silane) coated slides. Once mounted, the slides should be dried to remove any water that may be trapped under the section.

3. Deparaffinization
Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section. Keep the slides in the tap water until ready to perform antigen retrieval. At no time from this point onwards should the slides be allowed to dry. Drying out will cause non-specific antibody binding and therefore high background staining.

4. Antigen Retrieval
Most formalin-fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigenic sites. The two methods of antigen retrieval are heat-mediated (also known as heat-induced epitope retrieval, or HIER) and enzymatic. Both antigen retrieval methods serve to break the methylene bridges and expose the antigenic sites in order to allow the antibodies to bind.

5. Immunohistochemical Staining
It is highly recommended that researchers review the methodology and variations in IHC protocols (see also references at the end of this section).


A positive tissue control is strongly recommended to ensure that the antibody is performing as expected. Depending on the experiment, it may also be useful to include a negative tissue control: a tissue in which the protein of interest is not expected to be found.

To estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen (e.g. BrdU) having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control. For whole serum antibodies, use normal serum from an unimmunized animal of the same species as the primary antibody.

Signal amplification

To achieve a stonger signal, various strategies have been developed to add more enzyme or fluorophore to the target of interest.
a) Avidin-biotin complex (ABC)
b) Labeled streptavidin biotin (LSAB)
c) Tyramide signal enhancing (TSE)


IHC/ICC/IF - Troubleshooting

No staining

The primary antibody and the secondary antibody are not compatible
Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is raised in rabbit, use anti-rabbit secondary).

Not enough primary antibody is bound to the protein of interest
Use less dilute antibody. Incubate longer (e.g. overnight) at 4°C.

The antibody may not be suitable for IHC procedures which reveal the protein in its native (3D form)
Test the antibody in a native (non-denatured) WB to make sure it is not damaged.

The primary/secondary antibody/amplification kit may have lost its activity due to improper storage, improper dilution or extensive freezing/thawing
Run positive controls to ensure that the primary/secondary antibody is working properly.

The protein is not present in the tissue of interest
Run a positive control recommended by the supplier of the antibody.

The protein of interest is not abundantly present in the tissue
Use an amplification step to maximize the signal.

The secondary antibody was not stored in the dark
Always prevent the secondary antibody from exposure to light.

Deparaffinization may be insufficient
Deparaffinize sections longer, change the xylene.

Fixation procedures (using formalin and paraformaldehyde fixatives) may be modifying the epitope the antibody recognizes
Use antigen retrieval methods to unmask the epitope, fix for less time.

The protein is located in the nucleus and the antibody (nuclear protein) cannot penetrate the nucleus
Add a permeabilizing agent to the blocking buffer and antibody dilution buffer.

The PBS buffer is contaminated with bacteria that damage the phosphate groups on the protein of interest
Add 0.01% azide in the PBS antibody storage buffer or use fresh sterile PBS.


High background

Blocking of non-specific binding might be absent or insufficient
Increase the blocking incubation period and consider changing blocking agent.

The primary antibody concentration may be too high
Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but targeted binding is best).

Incubation temperature may be too high
Incubate sections or cells at 4°C.

The secondary antibody may be binding non-specifically (damaged)
Run a secondary control without primary antibody.

Tissue not washed enough, fixative still present
Wash extensively in PBS between all steps.

Endogenous peroxidases are active
Use enzyme inhibitors i.e. levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.

Fixation procedures (using formalin and paraformaldehyde fixatives) are too strong and modified the epitope the antibody recognizes
Change antigen retrieval method, decrease the incubation time with the antigen unmasking solution.

Too much amplification (amplification technique)
Reduce amplification incubation time and dilute the amplification kit.

Too much substrate was applied (enzymatic detection)
Reduce substrate incubation time.

The chromogen reacts with the PBS present in the cells/tissue (enzymatic detection)
Use Tris buffer to wash sections prior to incubating with the substrate, then wash sections/cells in Tris buffer.

Permeabilization has damaged the membrane and removed the membrane protein
Remove permeabilizing agent from your buffers.

Non-specific staining

Primary/secondary antibody concentration 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.

Endogenous peroxidases are active
Use enzyme inhibitors i.e. levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase. 

The primary antibody is raised against the same species as the tissue stained (e.g mouse primary antibody tested on mouse tissue). When the secondary antibody is applied it binds to all the tissue as it is raised against that species
Use a primary antibody raised against a different species than your tissue.

The sections/cells have dried out
Keep sections/cells at high humidity and do not let them dry out.


IHC/ICC/IF - References

Immunohistochemistry: Basics and Methods (1st Edition): I.B. Buchwalow & W. Böcker; Sprigner Verlag Heidelberg Berlin (2010)

Immunocytochemical Methods and Protocols (3rd Edition): C. Oliver & M.C. Jamur; Methods Mol. Biol. 588, 1-416 (2010)


IHC/ICC/IF - Online Resources





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