Tips and hints for your experiments

In an immunohistochemistry (IHC) experiment a primary antibody binds specifically to a protein of interest present in a tissue. The antibody binding is then visualized by a detection system, which provides information about if and where the protein is present in the tissue. IHC is a common method in diagnostics to determine morphological abnormalities and the presence of biomarkers indicative of certain diseases such as cancer.

Since the first IHC experiments in the 1940s much progress has been made in terms of developing different and increasingly sensitive detection systems. In the beginning almost exclusively secondary antibodies conjugated to enzymatic labels were used for visualization, while these days kits, directly conjugated primary antibodies and fluorescent labels are increasing in popularity. This trend is due to researchers wanting to detect several antigens simultaneously in one tissue specimen. This process is known as multiplexing and has many advantages, including cost and time savings and increased sample characterization.

Immunohistochemistry - Tips and hints for your experiments

Tips and hints for your IHC-paraffin experiments

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IHC staining of paraffin-embedded tissues (IHC-P)

Before starting the experiment, make sure you have selected an antibody that has been validated in IHC-P, that you have included controls in your experimental design, and that your microscope is set up to detect the antibody staining.

View a list of all IHC-paraffin tested antibodies.

  1. Mount and section samples
  2. Heat sections on the specimen slide to improve adherence
  3. Remove paraffin and rehydrate the tissue
  4. Perform heat induced or protease induced epitope retrieval (optional step; depending on tissue and protein of interest)
  5. Washing
  6. Block endogenous peroxidases, phosphatases (for enzymatic labels) and biotin (when using biotin/avidin systems)
  7. Perform PBS wash
  8. Block non-specific binding sites
  9. Perform PBS wash
  10. Incubate with primary antibody
  11. Perform 3 PBS washes
  12. Incubate with secondary antibody
  13. Perform 3 PBS washes
  14. Incubate with amplification reagent
  15. Perform 3 PBS washes
  16. Incubate with DAB or other substrate solution (for enzymatic labels only)
  17. Perform ddH2O wash
  18. Counterstain
  19. Dehydrate tissue sections (only needed when organic mounting media are used)
  20. Mount coverslip

Detailed protocols for IHC staining of frozen and paraffin embedded tissues can be found here.

Tips for step 4 – Perform heat induced or protease induced epitope retrieval

Antigens can be masked as a result of the fixation process, which makes antibody binding impossible. The unmasking can be reversed with a technique called antigen retrieval/antigen unmasking, which is either mediated by heat (HIER; heat-induced epitope retrieval) or proteases (PIER; proteolytic-induced epitope retrieval). The latter uses enzymes such as proteinase K, trypsin and pepsin. The PIER method acts by degrading the peptides masking the epitope. However, PIER might also result in alterations to the specimen morphology or the antigen itself. Consequently, PIER is less frequently used than HIER, which acts by restoring the secondary and tertiary structure of an epitope.

In order to establish whether antigen retrieval should be performed and by which method the following guidelines should be followed:

  • Perform a literature search to determine how other researchers have visualized your antigen of interest.
  • Check if the antibody supplier recommends a specific antigen retrieval protocol.
  • If no specific protocol is available, we recommend using a HIER rather than a PIER protocol.
  • For HIER always start with a neutral antigen retrieval buffer, such as Bio-Rad’s BUF025A and compare to a sample for which no antigen retrieval was performed.
  • If the neutral staining solution did not yield a good staining, alkaline or acidic antigen retrieval buffers should be tested 
  • In addition to pH, other parameters to be optimized are temperature and duration. Ideally try various pH, temperature and time combinations.
  • In order to exclude artifacts caused by the HIER process always include a control sample for which staining without the HIER step was performed.

Tips for step 6 – Block endogenous peroxidases, phosphatases and biotin

To avoid staining artifacts it is important:

  • to block endogenous peroxidases and phosphatases prior to using alkaline phosphatase (AP) / horseradish peroxidase (HRP) antibody conjugates. For blocking endogenous peroxidase activity Bio-Rad offers ready to use peroxide blocking reagent.
  • to block endogenous alkaline phosphatase activity levamisole is most commonly used.
  • to block endogenous biotin when using avidin/biotin or streptavidin/biotin detection systems. For this specific purpose Bio-Rad offers ready to use avidin-biotin blocking system.

Tips for step 8 – Block non-specific binding sites

Blocking should be performed prior to incubation with the primary antibody to prevent non-specific antibody binding.

  • Use normal serum from the same species as the one in which the secondary antibody was generated in.
  • Block with 10-20% normal serum.
  • Never block with normal serum from the same species as the primary antibody was generated in. This type of blocking serum on the one hand could lead to blocking of reactive sites and on the other hand to higher background.
  • If serum is unavailable, use bovine serum albumin, non-fat milk or gelatin. 

View all Bio-Rad sera here.

Tips for step 10 – Incubate with primary antibody 

  • Check on the manufacturer’s datasheet that the antibody has been tested in the specific immunohistochemical method intended to be used (e.g. IHC-paraffin). 
  • We recommend using a polyclonal antibody when establishing an IHC protocol. Although antigen retrieval is possible, the efficiency of the process is variable and certain epitopes might still remain inaccessible. Therefore a polyclonal antibody, which recognizes a multitude of epitopes due to its heterogeneous nature, provides a definite advantage over a monoclonal antibody which recognizes a single epitope.
  • Prior to performing the experiment check the localization of the antigen. To get an initial idea of the expected staining the relevant datasheet of the antibody supplier or web resources such as “The Human Protein Atlas” can be consulted.
  • When using an antibody for the first time always determine the optimal antibody dilution by performing staining with multiple antibody concentrations. This should be done for both the primary and secondary antibody.
  • To ensure that the observed staining is specific include IHC staining controls in your experimental design. Information on what IHC controls should be included can be found here.

Tips for step 12 and 14 – Incubate with secondary antibody and amplification reagent 

  • We recommend the use of directly conjugated antibodies in IHC only for the detection of very abundant target proteins (e.g. beta-actin and alpha-tubulin). 
  • For medium to low abundant proteins, we recommend using secondary antibodies for detection. We make this recommendation due to the fact that multiple secondary antibodies bind to a single primary antibody thereby leading to amplification of the signal.
  • For very low abundant proteins, we recommend using biotinylated secondary antibodies in combination with conjugated avidin/streptavidin,  Neutralite Avidin, and  Neutralite Avidin:Cy5 . We make this recommendation due to the high signal amplification of this method, which is due to a single avidin molecule being able to simultaneously bind up to four biotin molecules. 
  • When using biotinylated antibodies, ensure to block endogenous biotin prior to primary antibody incubation. For this specific purpose Bio-Rad offers a ready-to-use avidin-biotin blocking system.
  • For your convenience, we also offer the Histar Detection kit series, STAR3000, which provides linking and labeling reagents for the visualization of cellular antigens in tissue specimens. The Histar detection kits are biotin-free systems based on a novel polymer-based labeling technology. 
  • When selecting a fluorophore conjugated secondary antibody, ensure that your microscope is able to excite and detect the fluorophore appropriately. This can be done by looking up the excitation and emission data of the fluorophore of interest and the lasers/filters included in your microscope.
  • Select photostable fluorophores such as Alexa Fluor® and DyLight Fluor® dyes rather than FITC and PE, which are highly susceptible to fading/photobleaching.
  • When performing experiments with multiple fluorescent labels, ensure that each fluorophore can be spectrally separated and that one fluorophore does not get detected in another fluorophore’s channel (a process known as bleed-through). For this purpose we recommend to mock up the fluorophore excitation and emission spectra with the help of a spectrum viewer at the experimental design stage.

View all secondary antibodies 
View all avidin/streptavidin products

Tips for step 16 – Incubate with DAB or other substrate solution 

In immunohistochemistry the enzymatic labels horseradish peroxidase (HRP) and alkaline phosphatase (AP) are mainly used. 

In an immunoenzymatic staining a colored precipitate is formed due to the reaction of an enzyme with its substrate. This reaction converts a chemical compound called chromogen into the precipitate (see below formula; reference (1)). 

Factors to consider:

  • The precipitate color varies depending on the enzyme and chromogenic substrate combination. For example selecting DAB (3,3' Diaminobenzidine) as an HRP substrate leads to a brown staining while choosing AEC (3-Amino-9-Ethylcarbazole) results in a red one. 
  • When selecting chromogens do not simply select on staining color alone. Other factors to consider are the chromogen’s staining efficiency, staining intensity, and compatibility with organic mounting media (see mounting media section).
  • When designing experiments with multiple enzymatic labels care has to be taken that the final precipitate colors are spectrally differentiable (for example AP/Fast Red (red) and HRP/DAB (brown) is a good combination while AP/Fast Red (red) and HRP/AEC (red) is not).
  • Also take care taken that the counterstain does not have the same color as the precipitate (see counterstain section). 

The most popular chromogens for both HRP and AP and the resulting precipitate colors are shown below (adapted from (1)).

HRP substrate
Precipitate color
3,3'-Diaminobenzidine (DAB)
4-Chloro-1-Naphtol (CN)
P-Phenylenediamine Dihydrochloride/pyrocatechol (Hanker-Yates reagent)


AP substrate
Precipitate color
Fast Red TR
Bright red
New Fuchsin
Fast Blue BB



(1) Agilent Technologies (2009), Education Guide: Immunohistochemical staining methods edition 5.
Available here (Accessed: January 02, 2015)

Tips for step 18 – Counterstain

Like controls, counterstaining is crucial for every IHC experiment, as the counterstain provides background contrast and puts the observed staining into perspective (e.g. by visualizing nuclei).

  • Especially for multi-color experiments, care should be taken when selecting counterstains to ensure that they are spectrally differentiable from the color of the antibody staining (e.g. DRAQ5 should be used as a nuclear counterstain rather than DAPI when using antibodies conjugated to blue emitting fluorophores such as Alexa Fluor® 405 or DyLight® 405).
  • In the interest of time and for your convenience, we recommend using mounting media that contain counterstains.

The most commonly used chromogenic and fluorescent counterstains are shown below (adapted from (1)). 

Chromogenic counterstains
Counterstain for
Hematoxylin (4 types - Harris’s, Mayer’s, Carazzi’s, and Gill’s)
Fast red
Methylene blue
Methylene green
Toluidine blue


Fluorescent counterstains
Counterstain for
Hoechst 33258/33342
Propidium iodide
Sytox® Green
Wheat germ agglutinin (WGA)
Variable; depending on which dye was conjugated to WGA
Plasma membrane
Variable; depending on which dye was conjugated to phalloidin



(1)Paul, C., Counterstaining for Immunohistochemistry: Choices, Choices
Available here (Accessed: December 02, 2015).

Tips for step 20 – Mount coverslip

Mounting media are essential for making permanent slides and assist in making the coverslip adhere to the slide. Mounting has the purpose of protecting the specimen from damage while adding contrast during microscopy. Two types of mounting media exist:

  1. Aqueous (hydrophilic; examples of aqueous mounting media are glycerol and glycerol jelly)
  2. Organic solvent based (hydrophobic; examples of organic solvent based media include Euparal and Canada Balsam)

Mounting media can be further differentiated into media that solidify or stay liquid (2). In general organic solvent based media solidify, while aqueous ones remain in a liquid state.

Factors to consider:

  • Do not use organic mounting media for fluorescent labels. 
  • When using organic mounting media care should be taken as some precipitates, formed as the result of the chromogen substrate reaction, are alcohol soluble. One of those examples is the red precipitate formed as the result of the reaction of horseradish peroxidase (HRP) and 3-Amino-9-ethylcarbazole (AEC) (1).
  • For wide-field microscopy mounting media that solidify should be used.
  • For 3D imaging mounting media that stay liquid have to be used (3). When using mounting media that remain liquid, the coverslip edges must be sealed with nail polish to prevent the medium from leaking.
  • To prevent photobleaching (a process in which the chemical destruction of a fluorophore results in the loss of fluorescence) slides should be stored in the dark.
  • Further bleaching can be prevented by using mounting media containing antifade reagents. However as not every fluorophore is compatible with every antifade reagent; we recommended to check the manufacturer’s datasheet prior to use.

Bio-Rad offers permanent aqueous mounting medium. This mounting medium is suitable for use with peroxidase and alkaline phosphatase chromogens, Fast red, BCIP/NBT, BCIP/INT, Aminoethylycarbazole (AEC) and DAB/DAB with cobalt and nickel chromogens, and is also compatible Nuclear fast red (NFR) counterstains. 


(1) Renshaw, S. (editor) (2007) Imunohistochemistry. Methods Express Scion Publishing
(2) Kim, O., MicrobeHunter Microscopy Magazine, An overview of mounting media for microscopy.  Available here (Accessed: July 15, 2014).
(3) North, A.J. (2006) Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. JCB vol. 17 no. 1 9-18.

Alexa Fluor ® is a registered trademark of Molecular Probes Inc. OR, USA
DyLight ® is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries
DRAQ5 ™ is a trademark of Biostatus Limited.
Sytox Green ® is a trademark of Life Technologies Company