Immunohistochemistry

Immunohistochemistry
Tips and Tricks for your IHC-paraffin Experiments
IHC Problems? We Won’t Leave You Hanging Out to Dry

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Mouse liver paraffi IHC-Fr on mouse Mouse liver
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Over 1600 antibodies tested in IHC-frozen and IHC-paraffin

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Tips and Tricks for Your IHC-paraffin 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 secondary antibodies conjugated to enzymatic labels were almost exclusively 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
multi-plexing and has many advantages, including cost and time savings as well as increased sample characterization.

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.

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 (this is an optional step; depending on tissue and protein of interest)
5. Perform wash
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

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 epitope 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

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Tips and Tricks for Your
your IHC-paraffin experiments
Experiments

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 a ready-to-use peroxide blocking reagent
(BUF017B)
■■To block endogenous biotin when using avidin/biotin or streptavidin/biotin detection systems. For this specific purpose
Bio-Rad offers a ready-to-use avidin-biotin blocking system (BUF016)
■■ To block endogenous alkaline phosphatase activity levamisole is most commonly used

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 that the primary antibody was generated in. This type of blocking serum

could lead to blocking of reactive sites or higher background

■■ If serum is unavailable, use bovine serum albumin, non-fat milk or gelatin

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 first 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 other web resources 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

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 suggest using biotinylated secondary antibodies in combination with conjugated avidin/
streptavidin. 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 endogenous biotin is blocked prior to primary antibody incubation. For this specific
purpose Bio-Rad offers a ready-to-use avidin-biotin blocking system (BUF016)
■■
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 human 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

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Tips and Tricks for Your
your IHC-paraffin experiments
Experiments

When performing experiments with multiple fluorescent labels, ensure that each fluorophore can be spectrally separated. This
■■

ensures that one fluorophore does not get detected in another fluorophore’s channel (a process known as bleed-through). For this
purpose we recommend mocking up the fluorophore excitation and emission spectra with the help of a spectrum viewer at the
experimental design stage

Tips for step 16 – Incubate with DAB or other substrate

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; Agilent Technologies 2009a).

1. Enzyme (E) + Substrate (S) = ES complex (rather transient)

2. ES → E + Product (P)

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 so 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 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 Agilent
Technologies 2009b):
HRP Substrate Precipitate Color AP Substrate Precipitate Color

3,3’-Diaminobenzidine (DAB) Brown Fast Red TR Bright red

3-Amino-9-Ethylcarbazole (AEC) Rose-red New Fuchsin Red
4-Chloro-1-Naphthol (CN) Blue Fast Blue BB Blue
P-Phenylenediamine Dihydrochloride/ Blue-Black
pyrocatechol (Hanker-Yates reagent)

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® Fluor 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 Paul 2013):

Chromogenic Counterstains Color Counterstain for

Hematoxylin (4 types - Harris’s, Blue Nucleus
Mayer’s, Carazzi’s, and Gill’s)
Fast red Red Nucleus
Methylene blue Blue Nucleus
Methylene green Blue/green Nucleus
Toluidine blue Blue Nucleus
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Tips and Tricks for Your IHC-paraffin Experiments

Fluorescent Counterstains Color Counterstain for

DAPI Blue Nucleus

DRAQ5™ Red Nucleus
Hoechst 33258/33342 Blue Nucleus
Propidium iodide Blue/green Nucleus
®
Sytox Green Green Nucleus
Wheat germ agglutinin (WGA) Variable; depending Plasma membrane
on which dye was
conjugated to WGA
Phalloidin Variable; depending Filamentous
on which dye was
Actin
conjugated to WGA

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 (Kim no date). 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) (Renshaw 2007)
■■ For wide-field microscopy mounting media that solidify should be used

■■ For 3D imaging mounting media that stay liquid have to be used (North 2007). 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

References:
Agilent Technologies (2009a). Education Guide: Immunohistochemical staining methods edition 5.
http://www.dako.com/uk/08002_03aug09_ihc_guidebook_5th_edition_chapter_3.pdf, accessed November 02, 2015.
Agilent Technologies (2009b). Education Guide: Immunohistochemical staining methods edition 5.
http://www.dako.com/08002_03aug09_ihc_guidebook_5th_edition_chapter_15.pdf, accessed January 02, 2015.
Paul C (2013). Counterstaining for Immunohistochemistry: Choices, Choices. http://bitesizebio.com/13467/counterstaining-for-
immunohistochemistry-choices-choices/, accessed December 02, 2015.
Kim O. MicrobeHunter Microscopy Magazine, An overview of mounting media for microscopy.
http://www.microbehunter.com/an-overview-of-mounting-media-for-microscopy/, accessed December 02, 2015.
Renshaw S (editor) (2007). Imunohistochemistry (Methods Express Scion Publishing).
North A J (2006). Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. JCB vol. 17 no. 1 9-18.

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F4/80 (clone CI:A3-1): The Murine Macrophage of Choice

F4/80 CI:A3-1 is the definitive and most widely referenced antibody clone in mouse macrophage research, used in almost 1000
publications. Our F4/80 antibodies (MCA497) are performance guaranteed in a wide range of applications including flow cytometry,
immunofluorescence/immunocytochemistry, immunohistochemistry (IHC-frozen, paraffin and resin), immunoprecipitation and western
blotting.

To aid your research and understanding of this marker, a selection of F4/80 references are available, providing information about
detailed methods, uses and advantages of our F4/80 antibody. Further references can be found on CiteAb, where there are over 900
F4/80 clone CI:A3-1 citations.

As part of our immunohistochemistry protocol collection, we offer both protocols for HIER (Heat Induced Epitope Retrieval) and PIER
(Protease Induced Epitope Retrieval) mediated F4/80 epitope retrieval. Enzyme pre-treatment using proteinase K is recommended for
tissues fixed for 24 hours, while heat induced F4/80 retrieval using citrate buffer (pH 6.0) is recommended for tissues fixed for seven
days or more in neutral buffered formalin.

Standard HIER protocol

Reagent
Citrate buffer (10 mM citric acid, pH 6.0) Citric acid (anhydrous), 1.92 g
Distilled water, 1000 ml
Mix to dissolve
Adjust pH to 6.0 with 1 M NaOH (be sure to mix well)
Store this solution at room temperature for 3 months, or at 4oC for longer usage

Method
1. Dewax paraffin sections and rehydrate using preferred protocol
2. Pre-heat sodium citrate buffer in a staining vessel to 95-100°C
3. Immerse slides in the citrate buffer and incubate for 10 minutes at 95-100°C. Check the citrate buffer level, add more if
necessary, and then incubate for a further 10 minutes at 95-100°C
4. Allow sections to cool for 20 minutes
5. Rinse sections with PBS
6. O
 ptional step; if required proceed with endogenous peroxidase de-activation, Bio-Rad offers peroxide blocking reagent
(BUF017B)
7. Proceed with preferred staining protocol

In order to view all IHC-protocols, including PIER and IHC-frozen protocols (Figure 1), please go to
bio-rad-antibodies.com/protocols.

Figure 1: IHC-frozen mouse intestine

cryosections stained with anti-F4/80
(MCA497; red) and anti-PLVAP (MCA2539;
green) antibodies. PVLAP stained blood
vessel endothelium surrounds F4/80-labeled
macrophages in the lamina propria. Nuclei were
counterstained with DAPI.
F4/80

PLVAP Merged Image

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