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 bindto tissue components and reveal their presence.

Immunohistochemistry (IHC) (aka Immunohistology) provides data about expressionof 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, butdoes 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 sectionsare rehydrated.

Be aware that the fixation process can damage antigens, which can mean thatsome 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 littleantigen 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 backgroundstaining 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



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/

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 studyantigen 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-reactivityof secondary antibodies with endogenous mouse Ig


Direct staining in IHC?

The application of direct staining (i.e. where the primary antibodyis directly linked to either HRP or Alk Phos) has a number ofadvantages:-

Significant savings in time and reagents – no secondary antibodiesrequired 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



Add secondary antibody



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 commercialantibody, or of your own reagent, can be expensive and time consuming

Lightning-Link® conjugation kits fromInnova Biosciences offer a very quickand easy-to-use solution, that requiresas 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 simplebut 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 cancalculate how to use Lightning-Link for your application


Q&A session

Type in questions

Click to pop out the questions box


San Diego, CAJune 23-26, 2014 Booth 4069


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Lightning-Link® is a registered trademark of Innova BiosciencesDyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries

Date post:	16-Jul-2015
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An introduction to immunohistochemistry

Science