THE COMPLETE GUIDE TO

Immunohistochemistry

ptglab.com
 TABLE OF CONTENTS
Introduction to Immunohistochemistry . . . . . . . . . . . . . . . . . . . 4

General Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Materials and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Tissue Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Immunohistochemistry Staining Protocol . . . . . . . . . . . . . 10

Tissue Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Antigen Retrieval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Immunostaining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Signal Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Common Troubleshooting Tips . . . . . . . . . . . . . . . . . . . . . . . . . 29

Contact Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

ON THE COVER: Top left: IHC analysis of human liver tissue using CYC1
IHCeasy Kit (KHC0245). Bottom right: IHC analysis of human colon tissue
using Cytokeratin 19 antibody (10712-1-AP).

The Complete Guide to Immunohistochemistry 3

 INTRODUCTION
SECTION HEADER
TO IHC

Introduction to
IMMUNOHISTOCHEMISTRY
Immunohistochemistry (IHC) enables the visualization of proteins in tissue while
retaining its microstructure. It helps to demonstrate the exact position and
distribution of the protein of interest in the analysed tissue section. You can then
visualize direct comparisons between experimental or pathological states, e.g.
between healthy and diseased tissues. During an IHC experiment, the protein of
interest (antigen) is detected by the binding of a primary antibody. This antibody-
antigen interaction is then visualized via chromogenic or fluorescent detection using
a secondary antibody.

For an IHC experiment to provide the best results, several steps of the IHC
protocol may require optimization to ensure specific antibody binding and optimal
visualization of the target protein. An IHC protocol can be influenced by various
factors, thus we must find the best working conditions to get strong and specific
staining.

The following guide outlines the general IHC protocol, as well as some useful tips
and hints on how to optimize each step.

Factors to be considered when designing an IHC experiment:

IHC Factor Considerations

Sample type FFPE, frozen
Antigen Species, level of expression, subcellular location
Epitope Conformation, post-translational modification
Blocking Sera, BSA, commercial buffer, temperature, pH, dilution,
incubation time

Primary antibody Monoclonal vs Polyclonal

Secondary antibody Species, label type
Signal detection Chromogenic, fluorescent
Counterstaining Chromogenic, fluorescent
Analysis Microscope, software based analysis, evaluation by eye
Controls Reagent controls, antigen controls

4 The Complete Guide to Immunohistochemistry

 INTRODUCTION
SECTION HEADER
TO IHC

Overview of the IHC Workflow for FFPE Samples

Organism Paraffin Embedding

Tissue After Fixation Tissue Sectioning
Collection & Dehydration

Mounting Section
on Coated Slide
60% Alcohol 80% Alcohol 95% Alcohol 100% Alcohol Xylene
Deparaffinization & Rehydration

Blocking Primary Antibody

Antigen Incubation
Retrieval

Secondary Antibody
Incubation
Fluorescent Chromogenic
Detection Detection

Add DAB Chromogen Solution

Counterstain Counterstain with

with DAPI hemotoxylin

Image through Image through

fluoresence microscope bright-field microscope

The Complete Guide to Immunohistochemistry 5

 GENERAL
SECTIONPROTOCOLS
HEADER

General Protocols – Section 1:

MATERIALS AND EQUIPMENT
• Freshly collected tissue • Xylene
• Cryo-embedding media (e.g. OCT) • Ethanol
or paraffin
• Antigen retrieval buffer
• PBS
• Heating source
• Sucrose
• Serum/BSA
• 4% Paraformaldehyde (PFA)
• TBS
or acetone
• Primary antibody
• Microtome or Cryostat
• Secondary antibody
• Embedding station
• Diaminobenzidine tetrachloride (DAB)
• Glass slides
• Hematoxylin
• Coverslips
• Mounting media
• Refrigerator
• Microscope
• Incubator

6 The Complete Guide to Immunohistochemistry

 GENERAL
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General Protocols – Section 2:

TISSUE PREPARATION
Preparing fixed tissues for paraffin-embedded sectioning
Here we describe the procedure for preparing formalin-fixed paraffin-embedded
tissue (FFPE) samples.

• For animal tissues, it is highly recommended to perform perfusion before

dissection to remove blood from tissues to eliminate potential non-specific signals
caused by endogenous IgG. Perfuse with ice-cold PBS to remove blood, followed
by perfusion with 4% PFA until adequate fixation is achieved. Alternatively, dissect
the desired tissues (<3mm thick), wash with cold PBS, place into a cassette and fix
in 4% PFA or 10% formalin overnight at 4ºC (no longer than 24h). The adequate
volume of fixative for fixation by immersion is 50-100x of the sample size.
• Rinse with running tap water for 30 minutes.
• Dehydration
Move cassettes into 50% ethanol for 45 minutes.
Move cassettes into 70% ethanol for 45 minutes. (Tissues can be
left in 70% ethanol for long term storage, if required.)
Move cassettes into 80% ethanol for 45 minutes.
Move cassettes into 95% ethanol for 45 minutes. Repeat this step a second
time, using fresh 95% ethanol.
Move cassettes into 100% ethanol for 45 minutes. Repeat this step 2 more
times, using fresh 100% ethanol each time.
• Clearing
Move cassettes into Ethanol : xylene (1 : 1 v/v) for 30 minutes.
Move cassettes into 100% Xylene for 30 minutes. Repeat this step
a second time, using fresh 100% Xylene.
• Wax infiltration
Prepare beakers containing molten paraffin at 56-58ºC.
Immerse cassettes into paraffin wax for 60 minutes. Repeat this step
a second time, using fresh paraffin.

The Complete Guide to Immunohistochemistry 7

 GENERAL
SECTIONPROTOCOLS
HEADER

• Embedding
Move cassettes onto an embedding station.
Using a mold made of thick plastic or stainless steel, coat the bottom layer
of the mold with molten paraffin.
Place the tissues on top of the base wax orienting them as desired. Partially
cover with more paraffin. Place the cassette on top of the mold and completely
fill the mold.
Place mold with tissues on a cold plate to solidify. The paraffin will solidify
in 10-15 minutes.
Remove embedded tissues from the mold. Store at room temperature.

Sectioning tissues on a microtome

• Section paraffin blocks at the desired thickness (usually 4-5 µm) on a microtome
and transfer them to a 40°C water bath containing distilled water.
• Transfer the sections onto charged slides coated with gelatin or poly-L-lysine. Place
the slides in an oven to dry at 50-55ºC for 4 hours (or 37ºC for 6-8 hours)
and store slides at room temperature until ready for use.

Preparing snap-frozen tissues for post-sectioning fixation

Here we describe how to snap-freeze tissue, section tissue on a cryostat and then
postfix on a slide.

• Place the tissue block (freshly dissected, <5mm thick) onto a mold.
• Use cryo-embedding media (e.g. OCT) to fully cover the entire tissue block.
• Completely freeze the tissue on the base mold by submersion in liquid nitrogen.
• Store the frozen tissue block at -80°C until ready for sectioning. (See section on
frozen sectioning below.)
• Once sectioned, fix slides by incubating them in either ice-cold acetone (-20 ºC)
for 20 minutes or 4% paraformaldehyde (PFA).

Preparing fixed tissues for frozen sectioning

Below is a general protocol for fixing tissues before sectioning on the cryostat.
• When possible, perfusion fixation is recommended for achieving the best
tissue morphology and clear background. Perfuse with ice-cold PBS to remove
blood, followed by perfusion with 4% PFA until adequate fixation is achieved.
Alternatively, dissect the desired tissues and fix them in 4% PFA or 10% formalin
overnight (no longer than 24 hours). The adequate volume of fixative for fixation
by immersion is 50-100x of the sample size.
• Tissues should then be dehydrated for cryoprotection in fresh 20-30% sucrose,
for 16-48 hours at 4°C.

8 The Complete Guide to Immunohistochemistry

 GENERAL
SECTIONPROTOCOLS
HEADER

• Embed the tissues in OCT medium using a mold and freeze by submersion in
liquid nitrogen.
• Store the tissues at -80°C until ready for sectioning.

Sectioning tissues on a cryostat

Users must be fully trained and follow manufacturer guidelines before using the
cryostat. The optimal cryostat temperatures recommended for different tissue types
are displayed in the table below.
• Before sectioning, transfer the frozen tissue block to a cryostat at the
recommended temperature (e.g. -20°C) and allow the frozen tissue block to
equilibrate to the cryostat temperature (around 30 minutes).
• Section the frozen tissue block into a desired thickness. Use glass slides (we
recommend charged slides) designed for IHC to prevent tissue slipping.
• Once cut, allow sections to dry thoroughly at room temperature. Sections can be
stored in a sealed slide box at -80°C for later use. Storage might influence the
antigenic potential, which varies for each protein. Therefore, minimizing storage
time or using freshly prepared slides prior to staining is recommended.
• (If necessary) Fixation: after immediately removing from the freezer, incubate slides
with either ice-cold acetone (-20 ºC) or 4% PFA for 10-20 minutes. The end user
must determine optimal fixation conditions for each tissue type.

Recommended Cryostat Temperatures for Unfixed Tissues

Brain, liver and lymph node tissues -10ºC / -15ºC
Thyroid, spleen, kidney and muscle tissues -15ºC / -20ºC
Tissue containing fat -25ºC
Tissue containing plenty of fat -30ºC

The Complete Guide to Immunohistochemistry 9

 GENERAL
SECTIONPROTOCOLS
HEADER

General Protocols – Section 3:

IMMUNOHISTOCHEMISTRY
STAINING PROTOCOL
Below is an outline of a standard IHC protocol. Steps may need optimizing and
adapting to suit individual requirements and different tissue types. All steps are
carried out at room temperature unless stated otherwise.

Deparaffinizing and Rehydration (for FFPE sections only)

• Immerse slides in xylene for 10 minutes. Repeat this step in fresh xylene for 10 min.
• Rehydrate sections by sequentially incubating with 100%, 95%, 80% and 60%
ethanol for 5 minutes each.
• Rinse sections with distilled water three times for 1–3 minutes each.

Antigen Retrieval (optional)

• Transfer slides to a microwave-proof container or a beaker on an electric stove and
cover them with the recommended antigen retrieval buffer. If no antigen retrieval
buffer is suggested, try Tris-EDTA (pH 9) first. See the “Antigen Retrieval” section
for further information.
• Heat in the microwave on medium power for 10 minutes.
• Allow slides to cool in the antigen retrieval buffer for approximately 35 minutes.

Quenching and Blocking

• Rinse slides three times with 1X TBS for 3 minutes each.
• Incubate slides with 3% H2O2 solution (diluted in distilled water) for 10 minutes to
quench endogenous peroxidase activity. For FFPE samples, this step is often not
necessary. See the section on “Quenching” for more information.
• Rinse slides three times with 1X TBS for 3 minutes each.
• Prepare 1% BSA or 5% normal blocking serum in 1X TBS. The serum should be
derived from the same species in which the secondary antibody was raised. Block
the sections for 30-60 mins.

Primary antibody incubation

• Incubate sections with primary antibody diluted in 1X TBS for 1 hour, or
overnight at 4°C; the optimal antibody dilution ratio should be pre-determined
by experimentation. Set up negative controls by omitting the primary antibody
incubation step for one slide per experimental condition.
• Following primary antibody incubation, rinse slides three times with 1X TBS for 3
minutes each.

10 The Complete Guide to Immunohistochemistry

 GENERAL
SECTIONPROTOCOLS
HEADER

Chromogenic Signal Detection

• Apply sufficient horseradish peroxidase (HRP) labeled secondary antibody (we
recommend a Polymer-HRP conjugate for best results) and incubate for 30 minutes
at room temperature.
• Rinse slides three times with 1X TBS for 3 minutes each.
• Prepare an appropriate volume of the DAB chromogen solution based on
manufacturer guidelines. Apply the substrate carefully and incubate for 5-10
minutes until a brown colour develops.
• Rinse sections gently with distilled water.
• Signal enhancement (optional): Immerse slides in 0.5-1% CuSO4 for 5 minutes.

Hematoxylin counterstaining (optional)

• To stain nuclei, add a few drops of Hematoxylin and incubate for 3 minutes.
• Rinse slides gently with distilled water.
• Transfer slides into a 1% HCl and 99% ethanol solution for 10 seconds and then
rinse briefly in distilled water.

Fluorescent signal detection

• Add sufficient fluorophore labeled secondary and incubate at room temperature
for 30 minutes in the dark. Ensure all steps following this are done in the dark or
with minimal exposure to light.
• Optional: DAPI is an excellent nuclear counter stain. This can be added here or
use mounting media with DAPI.

Dehydration and mounting

If performing fluorescence IHC, dehydration is not necessary.
• Immerse slides sequentially into 60%, 80%, 95% and 100% ethanol baths
for 5 minutes each.
• Immerse slides in xylene for 5 minutes. Repeat this step in fresh xylene
for 5 minutes.
• Mount the sections with sufficient mounting media and cover with a cover slip.
Air-dry in a well-ventilated area (e.g. fume hood).

The Complete Guide to Immunohistochemistry 11

 Select the Right Kit for Your IHC Workflow

IHCeasy Kits IHC Prep & Detect Kits IHC Detect Kits
Target-specific, complete IHC Kits for developing complete IHC Kits for highly-sensitive
workflow kits containing pre- workflows using any primary chromogenic signal
optimized primary antibody antibody of choice detection

Antigen
Retrieval Buffer

Washing Buffer

Blocking Buffer

Quenching buffer

Primary Antibody
Dilution Buffer

Primary Antibody

Secondary
Antibody

Chromogen

Signal Enhancer

Counter Staining
Reagent

Mounting Media

Control Slide
(optional)

Step-by-Step
Protocol

ptglab.com
 TISSUE
SECTION
PREPARATION
HEADER

TISSUE PREPARATION
Correct processing of tissue samples prior to staining them with desired antibodies
plays a critical role in determining the success of an IHC experiment. Once tissues
are harvested it is important to fix or freeze them as soon as possible to prevent their
degradation. Tissues can be either fixed in formalin followed by their embedding
in paraffin (IHC-FFPE) or they can be frozen (IHC-Fr) followed by their fixation in
formaldehyde or alcohol before or after cryosectioning. While fixation helps preserve
tissue morphology and target antigenicity, embedding in a solid medium such as
paraffin provides support to the tissues during sectioning.

FFPE samples Frozen samples

Fixation Typically fixed with Can be fixed with formaldehyde
formaldehyde prior or alcohols either before or after
to embedding sectioning

Embedding Paraffin wax Cryogenic embedding medium,

e.g. OCT

Sectioning Microtome Cryostat

Advantages • Preserves structural • Preserves antigenicity

morphology of tissues
• Suitable for studying post-
• Easy to handle without translationally modified targets
damaging tissues
• Simpler and shorter protocol
• Tissue blocks can be stored
for several years.

Challenges • Can lead to masking • Tissue structure may be

of epitopes due to over- impacted by ice crystals.
fixation
• Thicker sections result in lower
• Lengthy and complex resolution and poor quality
protocol images.

The Complete Guide to Immunohistochemistry 13

 ANTIGEN
SECTIONRETRIEVAL
HEADER

ANTIGEN RETRIEVAL
Aldehyde-based fixatives such as paraformaldehyde or formalin act by creating
cross-links between proteins in the tissue. In some cases, these cross-links mask
epitopes, inhibiting antigen access by antibodies and resulting in weak or absent
staining. Fortunately, this negative fixation effect can be reversed by a process called
antigen retrieval. The type and method of antigen retrieval that works best depends
on the tissue type and primary antibody. Our product-specific protocols state which
method is best for each of our antibodies, validated using IHC.

Comparison of Different Antigen Retrieval Methods

Tris-EDTA Sodium Citrate

Protease K None
KRT20 mAb (60183-1-Ig), human colon tissue

Heat-induced epitope retrieval (HIER)

HIER is carried by heating the slides in a specific buffer for a certain period of time.
Different heat sources like a microwave, pressure cooker, water bath, or a hot plate
are normally used for this step.
If not specified by the antibody manufacturer, first try HIER with Tris-EDTA (pH 9)
buffer. If this does not produce optimal retrieval then it might be worth trying HIER
with Citrate buffer (pH 6). Determine the optimal antigen retrieval conditions for your
samples by seeing which buffer produces the best staining, further optimization can
be achieved by varying the length of the incubation.

14 The Complete Guide to Immunohistochemistry

 ANTIGEN
SECTIONRETRIEVAL
HEADER

HIER acts by using thermal energy to break down the cross-links that bind to the
surrounding proteins or peptides. Another mechanism by which HIER is thought to
act is through removing calcium ions from the site of protein cross-links, a theory
that is supported by the fact that some HIER buffers such as Tris-EDTA are calcium
chelators.

Comparison of different HIER buffers

HIER Sodium Citrate (pH6) buffer HIER Tris-EDTA (pH9) buffer

KRT20 mAb (60183-1-Ig), human urothelial carcinoma

General HIER protocol:

• First heat the buffer to around 95ºC (near boiling).
• Apply the pre-heated buffer to the slides and incubate for 10–30 minutes.
• Remove from the heat and let the slides cool in the buffer for a further 35 minutes.

Proteolytic-induced epitope retrieval (PIER)

Proteolytic-induced epitope retrieval (PIER) technique results in a degradation of
peptides to unmask the epitopes of interest. Trypsin and proteinase K are enzymes
typically used in PIER, which act by breaking protein cross-links, which unmasks the
hidden antigen and thus increases the staining intensity and specificity of the primary
antibody.

General PIER protocol

• Prepare the trypsin and pre-heat to 37ºC. Pipette the enzyme solution onto the
section.
• Place the slides in a humidified container and then into a 37ºC incubator.
• After 15 minutes, remove the slides from the incubator and transfer to a rack in a
container with tap water. Rinse in running water for 3 minutes.
Other commonly used enzymes are proteinase k, pepsin, trypsin, and pronase.

The Complete Guide to Immunohistochemistry 15

 ANTIGEN
SECTIONRETRIEVAL
HEADER

HIER PIER
Uses Most common, Good for difficult epitope recovery
gentle epitope retrieval Can damage tissues
pH and Buffer Tris-EDTA pH9 most used; pH 7.4; proteinase K, trypsin,
Citrate buffer pH6; or other pepsin, pronase
buffers at neutral pH
Temperature* 95ºC 37ºC
Incubation Time* ~20 minutes ~10–15 minutes
* Optimal conditions always have to be determined by each laboratory and in accordance with the specific product
information.

Comparison of HIER versus PIER

HIER in TRIS-EDTA buffer PIER in Protease K buffer

VWF pAb (27186-1-AP), human tonsilitis tissue

Buffers for heat-induced or

proteolytic-induced epitope retrieval

Protease K Buffer (pH 7.4)

Tris-EDTA Buffer (pH 9.0) Citrate Buffer (pH 6.0)

16 The Complete Guide to Immunohistochemistry

 SECTION
BLOCKING
HEADER

BLOCKING
Blocking is essential to prevent non-specific binding of the antibody or other
reagents to the tissue. Even though the antibody might be specific, it can also
show avidity for other epitopes. Antibody-antigen binding is affected by different
intermolecular forces (e.g. hydrophobic binding, ionic interactions) and these forces
can result in non-specific binding to endogenous molecules, serum proteins, or
others. Non-specific binding then prevents visualization of the antigen-antibody
binding of interest. Therefore, prior to incubation with the primary antibody, a
blocking step should be carried out to block the entire epitopes on the tissue
without damaging the desired epitope. Different commercial buffer systems are
available.

Serum/Protein Blocking
Serum (from the same species as the secondary antibody) or bovine serum albumin
(BSA) is commonly used for blocking. Sera and BSA can help to prevent non-specific
binding to the many hydrophobic side chains of proteins present in tissues. When
multiplexing, blocking serum of all the hosts of the secondary antibodies is required
(e.g. using normal goat and donkey serum). If BSA is used, the addition of 0.1-0.5%
Triton-X or Tween can act as an additional blocking reagent to prevent unspecific
binding.

Endogenous Enzyme Blocking (Quenching)

When using a horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated
antibody for detection, endogenous levels of the enzyme need to be blocked.
This typically applies to kidney, liver, intestine and lymph tissue. HRP is blocked
with buffers containing H2O2, and AP is blocked with buffers such as acetic acid or
Levamisole.
Note: With FFPE sections, quenching with H2O2 for HRP-DAB staining is not always necessary. When staining
a tissue with any antibody for the first time, leave out the quenching step and see if there is any noticeable
increase in background. H2O2 can be damaging to tissues if left on too long and adds further time and
complexity to the protocol. Therefore, omitting this step could be beneficial. Please note that this step is
imperative when using frozen sections as the endogenous enzymes are not deactivated.

The Complete Guide to Immunohistochemistry 17

 SECTION
BLOCKING
HEADER

Adverse Effects of Quenching

Quenched with 3% H2O2 Not quenched

YY1 mAb (66281-1-Ig), human colon cancer tissue

No Effects of Quenching

Quenched
with
3% H2O2

Not
quenched

No antibody GPX4 mAB (67763-1-Ig)

GPX4 mAb (67763-1-Ig), human kidney tissue

Endogenous Biotin Blocking

Endogenous biotin is found to be high especially in liver, kidney and brain tissue.
Biotin blocking is necessary if working with an avidin-biotin detection system.

18 The Complete Guide to Immunohistochemistry

 IMMUNOSTAINING
SECTION HEADER

IMMUNOSTAINING
Selecting and optimizing antibodies for IHC
High-quality antibodies are desirable for their high specificity and low cross-reactivity,
generating reliable and successful staining. When choosing a primary antibody for
IHC, there are certain factors that you should consider:
• Number of citations in the desired application(s)
• Consult the literature and antibody comparison resources.
• View the in-house validation data from the antibody manufacturer to ensure that
the antibody is validated in the correct application in native samples.

Selecting a primary antibody for IHC

When choosing a primary antibody, the question of clonality always comes up
regarding whether a monoclonal or polyclonal one is favorable for IHC. In general,
polyclonal antibodies are more often used in IHC, but this decision always depends
on the target of interest and sample type.

Advantages of polyclonal Advantages of monoclonal

antibodies in IHC: antibodies in IHC:
• Heterogenous population • Homogenous population
• Recognizes multiple epitopes • Specific for a single epitope
• Less sensitive to changes (pH, tissue, • Detecting a single protein with
buffer, protein confirmation), stable high affinity, even though it shares
detection sequence similarities to other proteins
• More likely to cross-react in rare • Good for long term projects, minimal
species lot-to-lot variability

Disadvantages of polyclonal Disadvantages of monoclonal

antibodies in IHC: antibodies in IHC:
• Not specific for one epitope, can lead • Sensitive to changes (pH, tissue, buffer,
to cross-reactivity with similar proteins protein confirmation)
• Possibility of lot-to-lot variability • Lower avidity can be an issue for
low expressed targets
• Less chance of cross-reactivity with rare
species

The Complete Guide to Immunohistochemistry 19

 IMMUNOSTAINING
SECTION HEADER

Optimizing a primary antibody for IHC

The optimal conditions for the primary antibody vary between experiments and
tissue type. Therefore, these factors require optimization to generate the best results
for each experiment.
Below are some general steps on how to optimize your primary antibody in IHC:
• Try different antibody dilutions based on manufacturer guidelines. Alternatively,
dilutions of 1 in 50 or 1 in 300 are good starting points for optimization. Make sure
you keep the temperature and incubation time the same. You may need to go
even lower to get the best staining.
• If you have specific staining, but high background signal, try varying incubation
time and temperature. Here, shorter incubations at room temperature are
recommended.
• If your antibody has a high-affinity antibody with a high concentration, try
incubating for a shorter time.
• If your antibody has a high affinity with a low concentration, try increasing the
incubation time and lowering the incubation temperature.
• Polyclonal antibodies in general can be used at a higher working dilution than
monoclonal antibodies, due to monoclonals often having higher specificity for the
target and being diluted at a higher concentration.

Optimization of antibody concentration

1:50 1:100 1:200 1:400 1:800

1:1600 1:3200 1:6400 1:12800 1:25600

CD68 mAb (66231-2-Ig), human tonsillitis tissue

OVER

9,000
Primary Antibodies
for IHC

20 The Complete Guide to Immunohistochemistry

 IMMUNOSTAINING

Selecting and optimizing secondary antibodies for IHC

Selecting the right secondary antibody for your needs is essential to help reduce the
signal-to-noise ratio, leading to better quality staining. Below are some of the points
to be aware of when choosing a secondary antibody:
• Subclass specificity: Polyclonal primary antibodies are mainly IgG isotypes. Primary
monoclonal antibodies are occasionally of a different isotype and therefore require
an isotype-specific secondary antibody. For example, mouse-derived antibodies
have four IgG subclasses: IgG1, IgG2a, IgG2b, and IgG3. To select an antibody
that targets all subclasses, ensure it is against the “heavy and light” (H+L) chain.
• Cross-adsorption: Secondary antibodies can go through an additional purification
step to reduce potential cross-reactions with other species. Here, the secondary
antibody solution is passed through different columns containing sera of different
species to filter out the non-specific secondary antibody.
• F(ab´)2 fragments: High background staining can be due to the presence of
Fc receptors in certain tissue or cells (lymph nodes, spleen, macrophages etc).
A whole antibody can bind to the Fc region, while an F(ab’)2 fragment does
not, ensuring higher specificity. F(ab’)2 fragments also allow for better tissue
penetration due to their smaller size.

IHC controls
To ensure that the observed staining pattern is specific and not due to any cross-
reactivity or non-specific binding, it is important to include control slides as part of
your experimental design. Controls must be run alongside the main experiment.

Reagent controls
Secondary only control • Reveals level of autofluorescence/
• Process the slide as normal but omit artefacts
the primary antibody. Isotype control
• Ensures signal detected is specific for • Replace the primary antibody with a
the target non-immune IgG of the same species,
• Test all fluorophores in isolation isotype and concentration as the
primary antibody.
Endogenous only control
• Ensures observed signal is specific of
• Process the slide as normal but omit FAB paratrope-epitope binding
the primary AND secondary antibody.

The Complete Guide to Immunohistochemistry 21

 IMMUNOSTAINING

Antigen controls • Ensures that the observed staining

pattern is due to specific signals
Positive control
• Use www.uniport.org or www.pax-bd.
• Control tissue that is known to express org to find endogenous negative
the protein of interest. controls in tissues.
• Confirms the protocol is correct • CRISPR knockouts or siRNA
• Validates negative results knockdown cell lines are good
negative controls.
Negative control
• Checking mRNA levels of target
• Control tissue that is known not to proteins can also serve as a good
express the protein of interest. negative control.

Save your precious reagents.

Use our Hydrophobic Pen
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22 The Complete Guide to Immunohistochemistry

 SIGNAL DETECTION

SIGNAL DETECTION
After incubation with a primary antibody, there are a few choices when it comes to
signal detection: chromogenic or fluorescent, and direct or indirect.

Chromogenic Detection
Chromogenic IHC antibodies commonly use conjugations with enzymes such
as horseradish peroxidase (HRP) or alkaline phosphatase. When these enzymes
encounter their substrate, they produce a dark precipitate, which can then be
visualized using a light microscope. DAB (3,3’-diaminobenzidine) is the substrate of
HRP and is the most used chromogen. DAB is insoluble, extremely stable, and heat
resistant, thus making it a good choice for many researchers.

Substrates and Colors for Chromogenic Staining

Enzyme Substrate – Color

DAB – Brown
DAB and Nickel – Black
HRP – Horseradish Peroxidase
AEC – Red
TMB – Blue
Fast Red – Red
AP – Alkaline Phosphatase
NBT and BCIP – Black to Purple

For detecting low-abundance targets, additional steps are often necessary for
amplifying the primary antibody signal. While methods like avidin-biotin complex
(ABC) detection or labeled streptavidin-biotin (LSAB) detection exploit the high-
affinity complex between biotin and avidin for signal amplification, polymer-based
amplification is biotin-free and utilizes a polymer backbone with multiple enzyme
molecules.

The Complete Guide to Immunohistochemistry 23

 SIGNAL DETECTION

Avidin-Biotin Complex (ABC) Detection

• Utilizes biotin labeled secondary antibodies
• Large avidin–biotin–enzyme complexes bind to biotinylated secondaries.
• Addition of chromogenic enzyme substrate leads to signal amplification due to the
presence of large multi-enzyme complexes.
Advantages: Highly sensitive
Limitations:
• High background resulting from the presence of endogenous biotin
• Large size of the ABC complex may hinder tissue penetration.

Biotin
Avidin
Reporter Enzyme

Biolinylated
Secondary Antibody

Primary Antibody

Tissue Antigen

Labeled Streptavidin-Biotin (LSAB) Detection

• Used instead of ABC detection whenever avidin-biotin-enzyme complexes are
too large to penetrate target tissues
• Utilizes a streptavidin-enzyme conjugate
Advantages:
• 10X times more sensitive than the ABC method
• Less background and better tissue penetration
Limitations:
• High background resulting from the presence of endogenous biotin

24 The Complete Guide to Immunohistochemistry

 SIGNAL DETECTION

Biotin
Streptavidin
Reporter Enzyme

Biolinylated
Secondary Antibody

Primary Antibody

Tissue Antigen

Polymer-based Detection
• Preferred method of detection when endogenous biotin, present in some tissues,
leads to high background with the ABC or LSAB methods
• Utilizes secondary antibodies directly conjugated to a polymer backbone
containing multiple enzyme molecules
Advantages
• More sensitive compared to the ABC and LSAB methods
• Shorter protocol compared to the ABC and LSAB methods
Limitations
• May hinder tissue penetration due to the larger size of the polymer backbone

Reporter Enzyme

Dextran Backbone

Biolinylated
Secondary Antibody

Primary Antibody

Tissue Antigen

The Complete Guide to Immunohistochemistry 25

 SIGNAL DETECTION

Fluorescence Detection
As an alternative to enzymatic detection, fluorescent signal detection during IHC can
be performed using fluorophore-conjugated antibodies. The basic premise behind
fluorescence detection is that the conjugated fluorophore is excited by a certain
wavelength of light, which then undergoes a change in conformation, resulting in a
lowering of energy state called the “Stokes shift.” Light is then is emitted at a longer
wavelength. Specialized fluorophores emit distinct excitation/emission spectra,
meaning that you can image multiple target proteins in the same sample using
different detection fluorophores, which is called multiplexing. Multiplexing can be
done by using primary antibodies that are raised in different hosts, and can thus be
targeted by different secondaries. Alternatively, this can be performed using directly
conjugated primary antibodies (see the section below).

Labeling Steps: a 1 b 2 c 3

Primary Antibody
1 2 3
a Rabbit-anti-antigen 1
b Mouse-anti-antigen 2
c Goat-anti-antigen 3

a b c
Secondary Antibody
1 Donkey-anti-rabbit A488
2 Donkey-anti-mouse A546
3 Chicken-anti-goat A594
Antigen 1 Antigen 2 Antigen 3

Chromogenic vs Fluorescent Signal Detection

Fluorescence Chromogenic
Excellent Multiplexing Greater sensitivity

Advantages Target Co-localization Longer lasting signal

Higher Dynamic Range Easier imaging

Staining will fade over time Poor multiplexing/co-localization

Limitations Lower sensitivity Low dynamic range

More complex imaging Longer protocol

26 The Complete Guide to Immunohistochemistry

 SIGNAL DETECTION

Indirect vs Direct Detection

Direct detection is an approach where a primary antibody is directly conjugated with
an enzyme or a fluorophore, whereas indirect detection uses a labeled secondary
antibody. The choice of the detection method depends on the expression level of
the antigen. Directly labeled primary antibodies have slightly lower signal generation
and are therefore best suited for highly expressed antigens. Direct detection is
also advantageous for multiplexing experiments that analyze multiple protein
targets simultaneously in the same sample, as this negates the need to find primary
antibodies raised in different species. Medium-expressed antigens show the best
signal when analyzed via a secondary labeled antibody, as this helps to amplify the
signal intensity. For very low expressed proteins, indirect detection plus an enhancer
(e.g. streptavidin or a polymer) helps to further amplify the signal.

Indirect Direct

Fluorochrome
Fluorochrome
Secondary
Antibody
Primary
Antibody

Primary
Antibody Antigen Antigen

Indirect Staining

IF analysis of human kidney tissue using PTPRO antibody (67000-1-Ig, Clone: 2F2B4 ) and CoraLite594-Conjugated AffiniPure Goat
Anti-Mouse IgG(H+L), ACE2 antibody (21115-1-AP, green) and CoraLite488-Conjugated AffiniPure Goat Anti-Rabbit IgG(H+L).

The Complete Guide to Immunohistochemistry 27

 SIGNAL
SECTION
DETECTION
HEADER

Direct Staining

IF analysis of human lung cancer tissue using CoraLite®️ Plus 488 CD3 antibody (CL488-60181, Clone: 3F3A1 ), CoraLite®️555
pan-keratin antibody (CL555-26411, orange), and CoraLite®️ Plus 647 CD20 antibody (CL647-60271, Clone: 4A7G3, Magenta).

GO DIRECT WITH

CoraLite®
Fluorescent Dye-Conjugated Antibodies

28 The Complete Guide to Immunohistochemistry

 TROUBLESHOOTING
SECTION HEADERTIPS

Common
TROUBLESHOOTING TIPS
No/Weak Staining
POTENTIAL CAUSE SUGGESTED SOLUTION

Primary/secondary Use new lot of antibody.

antibody lost activity Improper storage – follow manufacturer’s instructions.
Antibody conditions Titrate antibody concentrations.
not fully optimized Adjust incubation times.
Protein of interest not
Run a positive control.
expressed
Protein of interest has
Use signal amplification.
low expression

Damaged epitope Try a different epitope retrieval technique.

Background staining / Non-specific staining

POTENTIAL CAUSE SUGGESTED SOLUTION

Primary/secondary Titrate antibody concentrations.

concentration too high Decrease incubation times.
Non-specific Prolong blocking step.
antibody binding Increase concentration of blocking solution.
Repeat or prolong washing step.
Poor sample washing
Increase concentration of blocking solution.
Damaged epitope Try a different epitope retrieval technique.
Endogenous enzymes
Perform a quenching step.
or biotin/lectin

The Complete Guide to Immunohistochemistry 29

 TROUBLESHOOTING
SECTION HEADERTIPS

Inappropriate cell morphology

POTENTIAL CAUSE SUGGESTED SOLUTION

Harsh antigen Optimize buffers, temperature, pH,

retrieval conditions incubation time, concentration.
Folded or torn tissue Optimize thickness of tissue slides.
sections Re-cut sections using sharper blade.
Under-fixation. Change fixative or fixation time.
Damaged epitope Ice crystals can sometimes damage morphology during
freezing. Try using FFPE tissues.

Uneven staining
POTENTIAL CAUSE SUGGESTED SOLUTION

Basic slides or out

Used charged slides.
of date slides
Avoid drying of tissue sections during
Dried out sections
the staining procedure.
Fix tissues immediately after collection
Delay in fixing tissues
to avoid antigen diffusion.
Incomplete
Use fresh xylene.
deparaffinization
Distilled water Use PBS wash buffer instead of distilled water.
loosens sections Do not pipette wash buffer directly on tissues.

Issues with Avoid using protein-based section adhesives on

section adhesion charged slides to prevent inconsistent adhesion.

30 The Complete Guide to Immunohistochemistry

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