An introduction to immunohistochemistry

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Welcome to our 14th webinar
An introduction to Immunohistochemistry:
Basic principles and how to simplify your
staining procedures
Dr Andy Lane

Dr Andy Lane
• What is immunohistochemistry?
• Tissue preparation and fixation
• Tissue pre-treatment
• Principles of immunostaining
• Staining protocols
• Choosing and using antibodies
• Troubleshooting
• Some special challenges
• Direct staining in IHC

Q&A session
Type in questions
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What is Immunohistochemistry?
Definition:
The microscopic study of tissues with the aid of antibodies that bind
to tissue components and reveal their presence.
Immunohistochemistry (IHC) (aka Immunohistology) provides data about expression
of antigens within the context of tissue structure. This offers some important
advantages over techniques such as Western blotting and flow cytometry.
CD34 antigen staining
of human colon

Tissue preparation
The best option to maintain cellular structure, but
does have a number of issues especially in relation to IHC.
Tissues needs to be fixed in formalin immediately after harvesting – generally
for 4-24 hours. Over-fixation should be avoided.
Following fixation tissues are dehydrated and then embedded in hot paraffin
(although plastic is sometimes used). Tissue blocks can be stored for many years.
5-10µm sections are prepared by microtome. Prior to labelling tissue sections
are rehydrated.
Be aware that the fixation process can damage antigens, which can mean that
some antibodies will not recognise their antigens.
Paraffin-embedded tissue

Tissue preparation
Small pieces of tissue can be snap-frozen in liquid nitrogen or isopentane.
Sections can then be cut using a cryostat, which keeps tissue frozen during processing.
Fixation is then carried out immediately before staining, often using acetone.
Tissue morphology is generally not so good with frozen sections, but there is little
antigen damage and most antibodies will react as expected.
Frozen tissue

Tissue pre-treatment
Formalin fixation can damage antigens due to cross-linking, which can mean
that antibodies no longer recognise the antigen. Two general “antigen-retrieval”
techniques are used to try to overcome this – proteolytic digestion and
heat-induced epitope retrieval (HIER).
Proteolytic digestion uses incubation of tissue sections in either trypsin or pronase.
Some antigens respond better to one enzyme than the other.
HIER may use either a pressure cooker, or commonly nowadays a microwave.
There tends to be three cycles of heating, either in an acidic buffer (pH6) or
alkaline buffer (pH9). Again, some antigens respond better to one pH than the other.
Paraffin-embedded tissue – antigen retrieval techniques

Tissue pre-treatment
Peroxidase
A number of tissues contain endogenous peroxidase which can lead to background
staining when using the HRP system. Blocking by pre-treatment with 0.3% hydrogen
peroxide in PBS is recommended.
Alkaline phosphatase
Similarly, some tissues contain endogenous AP. This can be blocked using a
pre-treatment with 5mM levamisole.
FFPE tissues tend to have much lowers levels of endogenous enzyme activity, so
blocking is most commonly required for frozen tissues.
Frozen tissues - endogenous enzyme inhibition

Principles of immunostaining

Basic staining protocol
Prepare tissue sections
and undertake any
antigen retrieval or
endogenous enzyme
inhibition
Block with 1-2%
species serum (same
as secondary antibody)
Add primary antibody
Incubate 30-60
minutes
Wash (3 x 5 minutes)
Add secondary
antibody
Incubate 30-60
minutes
Add substrate Wash Counterstain

Choosing primary antibodies
Specificity and characterisation
Validation in IHC?
If using in FFPE is there information on
requirement for antigen retrieval?
Check for supplier data, and if possible published
references
On-line resources may be available with data
http://www.proteinatlas.org/

Choosing secondary antibodies
Specific for the primary antibody!
Not all secondary antibodies are the same…….
consider epitope recognised, purification,
absorption etc.
Enzyme conjugate (HRP or Alk Phos) or
avidin/biotin system
Consider staining kits supplied by a number of
suppliers

Troubleshooting
No staining / weak staining
Primary antibody performance - check titre, correct retrieval methods
Secondary antibody performance - optimise titre, specificity
Consider antigen expression – does your tissue have this antigen?
Confirm substrate activity
Optimise incubation times and temperatures
Appropriate use of positive controls
Optimal tissue processing – poor fixation can damage antigens

Troubleshooting
High background staining
Are endogenous enzymes and/or biotin blocked effectively?
Primary antibody performance – is specificity as expected? Optimise titration
Secondary antibody cross-reactivity – effective serum blocking, titration
Optimise incubation times and temperatures for primary
and secondary antibodies
Increase washing steps
Optimal tissue processing – poor fixation can cause background staining

Special challenges – dual staining
Staining of two antigens simultaneously can be a very powerful technique to study
antigen expression, but presents particular challenges in IHC as indirect staining is
so commonly used.
Different primary antibody species (or sub-classes) are required, and
highly specific secondary antibodies with no cross-reactivity are vital


Special challenges – mouse on mouse
A large amount of basic research is carried out in mouse models, and using
mouse monoclonals for IHC staining is very challenging due to cross-reactivity
of secondary antibodies with endogenous mouse Ig

Direct staining in IHC?
The application of direct staining (i.e. where the primary antibody
is directly linked to either HRP or Alk Phos) has a number of
advantages:-
Significant savings in time and reagents – no secondary antibodies
required and fewer wash steps
Simple dual staining
Simple mouse on mouse staining
However, availability of directly conjugated antibodies for
IHC staining is highly restricted

Direct staining protocol
Prepare tissue sections
and undertake any
antigen retrieval or
endogenous enzyme
inhibition
Block with 1-2%
species serum (same
as secondary antibody)
Add primary antibody
Incubate 30-60
minutes
Add secondary
antibody
Incubate 30-60
minutes
Add substrate Wash Counterstain

Anti-human CD20 (clone L26) was conjugated to HRP using Lightning-Link®
kits, and used in IHC in comparison to a traditional indirect technique
Direct staining in IHC
Unconjugated L26, visualised with Dako EnVision HRP conjugated L26, 1/100
Samples shown are formalin-fixed, paraffin-embedded human bone marrow. CD20 is a B cell specific marker

Anti-human CD20 (clone L26) and anti-human CD3 (Clone CD3-12) were
conjugated to HRP and Alk Phos respectively using Lightning-Link® kits, and
used to stain FFPE human tonsil.
Direct staining in IHC – two colour staining

What if the antibody conjugate I need isn’t available?
The great majority of commercially available antibodies are not available
directly conjugated to HRP or Alkaline Phosphatase for IHC use.
Having a custom made conjugate prepared, either of a commercial
antibody, or of your own reagent, can be expensive and time consuming
Lightning-Link® conjugation kits from
Innova Biosciences offer a very quick
and easy-to-use solution, that requires
as little as 10µg of antibody.
Preparing direct antibody conjugates

What is Lightning-Link® technology?
The worlds fastest, easiest to use and most efficient conjugation technology!
• Only 30 seconds hands-on time!
• Over 50 labels available including:
Enzymes, fluorescent proteins / dyes, tandems, biotin & streptavidin
Lightning-Link®
Antibodies – Proteins – Peptides
Fast – Easy-to-use – Reliable

What is Lightning-Link® technology?
• 100% antibody recovery
• Fully scalable from R&D (10µg<) to Production / Manufacture (>1g)
• Virtually eliminates batch to batch variability
• Coupling of the label to antibodies, proteins or other biomolecules
• Covalent conjugation ensures long term stability
Lightning-Link®
Lightning Link® kits are available for conjugating antibodies to
HRP and Alkaline Phosphatase, as well as to Biotin and to a wide
range of fluorescent dyes

Conjugation considerations
You need to know some things about your reagent.
Lightning-Link conjugations are really simple
but you need protein in the right format to work effectively.
Concentration – 1mg/ml or higher is preferred
Purity – ensure other proteins have been removed,
and also make sure they haven’t been put back again afterwards!
Buffer formulation – most common formulations are suitable,
but ensure that amines such as glycine are truly absent,
as well as thiols such as DTT or mercaptoethanol. Tris is OK up to 20mM
Lightning Link kits are optimised for antibody labelling, but can easily be
adjusted to label other proteins. If you know the size of your protein you can
calculate how to use Lightning-Link for your application

San Diego, CA
June 23-26, 2014 Booth 4069

Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
Please keep an eye out for our future webinars and other exciting news on our
website and social media channels:
www.innovabiosciences.com/innova/webinars.html
YouTube: www.youtube.com/InnovaBiosciences

Innova Biosciences Ltd.
Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova Biosciences
DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries

An introduction to immunohistochemistry

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