Instructions for Viewers

• To share webinar via social media:

• To see speaker biographies, click:  View Bio under speaker name

• To ask a question, click the Ask A Question button under the slide window

• To share webinar via e‐mail:

Improving tissue-sample profilingThe optimization and application of immunohistochemistry

July 7, 2015

Webinar Series

Sponsored by:

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office Nissi M. Varki, M.D.

University of California San DiegoLa Jolla, CA

Kevin D. Long, Ph.D.EMD MilliporeTemecula, CA

Webinar Series

Improving tissue-sample profilingThe optimization and application of immunohistochemistry

July 7, 2015

Sponsored by:

IMMUNOHISTOPATHOLOGYImproving tissue-sample profiling: The optimization and

application of immunohistochemistry

Nissi Varki M.D.

Professor of Pathology

Director of Histopathology and Immunohistochemistry and

Mouse Phenotyping Shared Resources

Mice

Human

Comparative pathology and models of human disease

Methods for human IHC and histopathology can be used for comparative pathology and translational studies

Influenza receptor expression on tracheal epithelium differs between species

Monica Cheriyan, Pascal Gagneux, Dan Anderson, Harold MacClure, Ajit Varki & Nissi Varki

Red arrows:apical edge of tracheal epithelium

Human Chimp

Mouse GorillaGreen arrows: goblet cells

Gagneux et al. J.Biol.Chem. 278: 48245–48250, 2003

Human Heart has more capillaries

A possible explanation as to why humans are able to sustain effort and run long distances

Heart disease is caused by different pathological processes in humans versus great apes

Evolutionary Applications2:101-112, 2009

•Athymic nude mice are used as a model for cancer research

•Balb/c mice are used for hybridoma production

•Became a “mouse pathologist”

Using mouse models to study human disease

Mouse lungs must be inflated before microscopic examination

OCT infiltrated lung

Frozen section

Good morphology

Non-OCT infiltrated lung

Frozen section

Poor morphology

Mouse lungs must be inflated before use in histopathology

Mucin secreting cells are not present in healthy mouse lung

Mouse models of

asthma induce

inflammation

Inflammation induces mucus

production in mouse

lungs

Human Spleen Mouse Spleen

Mouse organs must be properly processed

Mouse heart has high endogenous fluorescence

Use of enzyme-labeled detection systems is preferred for IHC on certain organs in mouse

models of human disease

IMMUNOHISTOCHEMISTRY (IHC) ASSAYS

IMMUNO: use of antigen/antibody reagents

HISTO: histology sections, viewed using brightfield microscopy

CHEMISTRY: substrates used to detect bound epitopes, produce precipitates visible with brightfield microscopy

FLUORESCENCE: can be used where no substrate is needed, viewed using epi-fluorescence microscopy with appropriate

excitation and barrier filters

Section of mouse lung showing inflammation•Nonspecific binding causes background issues

•Should not be used for IHC assays

Always check tissue morphology prior to IHC

IHC assays are performed using two sample types

Cells grown, spun into a pellet, frozen or paraffin embedded and sectioned

Cells grown as a monolayerOR

Sections from tissues contain many different kinds of cells as well as extra-cellular matrix components

Control cells on slides

OR tissue sections from either frozen blocks or FFPE (paraffin blocks)

IHC example: cell pellet control and tissue sections on the same slide

Positive Control Negative Control Tissue

One slide, same antibody

Fixation can mask epitopes and interfere with detection

Paraformaldehyde fixed

Fixative effects on preserving epitopes in frozen sections

If the tissue is fixed for longer than

24 hours and paraffin embedded,

some mouse monoclonals may not

recognize the epitope, in spite of

using retrieval techniques

Acetone fixed

Third reagent: To detect primary and secondary labels • Fluorescence: FITC, CY2, CY3, PE, AMCA, or Alexa dyes• Enzymes: horseradish peroxidase or alkaline phosphatase

Secondary reagent: Can be labeled, not labeled, or conjugated with biotin or digoxygenin

Primary antibody: Can be conjugated for direct assays or not labeled for detection using other reagents

Tissue section: frozen or paraffin

Three basic steps for IHC assays

When testing a new antibody, one needs to know:

Species of origin of the primary antibody: Mouse, rabbit, rat, hamster, chicken, goat, sheep, or horse

In order to:–design secondary and tertiary detecting reagents–design reagents to block non-specific binding–design which cells or tissues will be used as

positive and negative controls

Design controls for optimizing binding of primary antibody

Tissue or cells to be tested can be:–human–mouse–rat–other

If the primary antibody is POLYCLONAL rabbit anti-x or goat anti-x

If the primary antibody is MOUSE monoclonalTissue or cells to be tested can be:

–human–mouse–rat–rabbit–other

Primary antibody: maybe conjugated or not

Tissue section: frozen or paraffin If the primary antibody is

RAT monoclonalTissue or cells to be tested can be:

–human–mouse–rat–rabbit–other

Design negative and positive controls for every IHC

A. Reagent controls include:•Control slide which receives dilution buffer alone (secondary reagent control)

•Control slide which receives IgG at the same concentration as the test antibody

•Control slide which receives another antibody which will highlight specific tissue elements, such as stroma

B. Tissue/cells controls include: •Tissue or section from a cell pellet, not expected to be positive•Tissue or section from a cell pellet, not expected to be positive•Blocking agent, pre-incubated with specific antibody to remove reactivity

Block non-specific binding to collagen

Reagents to block non-specific binding

If using horse radish peroxidase detection, block endogenous peroxidases:

•Treat Frozen sections for 30 minutes with 0.03% H2O2 in buffer

•Treat Paraffin sections for 30 minutes with 0.3% H2O2 in buffer

If using biotinylated reagents, block endogenous biotin:•Always block non-specific collagen binding sites with diluting

buffer

Note: Frozen sections of small intestine, placenta etc. do contain endogenous alkaline phosphatase so these tissues should not receive alkaline phosphatase conjugated reagents

Enzyme conjugates allow visible detection and evaluation of morphologyFluorescent conjugates allow more sensitive detection methods

•Remove endogenous peroxidases•Block endogenous biotin•Block for endogenous alkaline phosphatase,

if needed•Develop with insoluble substrates•Different colors for peroxidase/alkaline phosphatase•Counterstain nuclei with contrasting colors

Enhance detection using:

•Biotin/streptavidin

•Tyramideamplification

Deparaffinize and block non-specific sites

Frozen sections

Immunohistochemistry optimization

Paraffin sections

airdry

Acetone fix

Fix in formalin after blocking

Tissue section: Frozen or deparaffinized

Tertiary reagent label may be an enzyme or fluoresceinated

fluoresceinated compounds

Remove endogenous non-specific binding sites

CY2 , FITC

AMCA

PE, CY3

Horse Radish Peroxidase

Alkaline Phosphatase

Substrates:

DAB, AEC, red , SG, VIP

Substrates

•Vector Blue

•Vector Red

Primary

Secondary

TertiaryAlexa dyes

•10 mM citrate buffer pH 6, heat induced antigen retrieval

•1 mM EDTA pH 8, heat induced antigen retrieval

•Tris buffer pH 10, heat induced antigen retrieval

•Proteinase K treat for 10 minutes at room temperature

•Trypsin treat for 10 minutes at room temperature

•Other methods

Antigen retrieval on paraffin sections facilitates detection of hidden epitopes

Biotinyl tyramide uses the HRP enzyme

to deposit many biotin molecules that “cover”

the antigen antibody complexes

already formed over the epitope on the tissue section

Further amplification can be used to detect low abundance epitopes in tissue

Tissue section: deParaffinized

enhancing detection levels by a factor of 100-1000

Using double stain methods to determine if two antibodies

recognize the same epitope on cells in a tissue section

Caveats:

•Use lower dilutions of antibodies for double staining to compensate for additive effect

•Use antibodies sequentially and detect with different fluoresceinated tags, so same epitope will be recognized with a new color

•One antibody may bind to the less abundant but similar epitope

•One antibody may bind with higher affinity or recognize a more abundant epitope

Co-localization and detection of similar epitopes on the same tissue section, using fluorescent markers

IgG control

Program of Excellence in the GlycoSciences

http://pegnac.sdsc.edu/ Glycobiology Research and Training Center

http://grtc.ucsd.edu/

Acknowledgements

UC San Diego histology cores:

http://mousepheno.ucsd.edu/overview.shtml

https://cancer.ucsd.edu/research-training/shared-resources/biorepository/Pages/histology-and-immunohistochemistry.aspx

Funding agencies: National Institutes of Health

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office Nissi M. Varki, M.D.

University of California San DiegoLa Jolla, CA

Kevin D. Long, Ph.D.EMD MilliporeTemecula, CA

Webinar Series

Improving tissue-sample profilingThe optimization and application of immunohistochemistry

July 7, 2015

Sponsored by:

The art of IHC: Know your antibody, know your tissue, and you will always succeed Dr. Kevin Long / EMD Millipore

Science Webinar

July 7, 2015

IHC is like a battle of two opposing forces: the antibody and the tissue…

Know your own strengths and you will win half the battles

Know your enemies’ strengths and you will win half the battles

Know the nature of your target protein epitope, and your antibody design

Know the effects of tissue processing on your antibody penetration and binding

Know your strengths and those of your enemy and you will win all the battles

Antibody validation combined with tissue processing and pre-experiment optimization will yield high success in IHC!

What you will learn

Pre-experiment IHC planning:

Antibody design considerations

Target and tissue considerations

The value of pre-experiment optimization

Antibody design considerations

Antibodies have the unique ability to bind to regions of a molecule tightly and extremely specifically

These complex, mostly biologically made “probes” continue to be the most effective way to find and isolate a target, or identify cell or tissue types

Antibody specificity

Although it is true that antibody binding to a specific amino acid sequence or conformational form can be theoreticallymodeled and is predictable in theory

Decades of user experience shows that many factors may contribute to issues of cross-reactivity and nonspecific binding.

Antigenicity Uniqueness of the immunogen sequence Antibody concentration Buffers used Immunization Sample preparation techniques

Antibody specificity does not mean reliability or ease of use

Reviewer comment: The buffer chosen to dilute the antibody used in this study has detergents, which are known to affect the HRP/DAB chromogenic reaction.

Know & justify the steps in your protocol Avoid ‘one size fits all’ protocols Every antibody is different so user MUST

optimize for each antibody AND lot

Antibody choice can depend on your application

• Target considerations• Sample considerations• Sample prep considerations• Ab design considerations

Target antigen considerations affect assay designGood forethought in experimental design is fundamental to choosing the components of your immunoassay.

Considerations for the target antigen: • Internal?• External?• Transmembrane?• Secreted? • Tertiary native conformation important?• Quaternary structure subunit binding important?• Are you trying to detect, measure, localize, isolate, or a combination

of these?

Determine the type of sample being tested

Key Considerations: Nature of your target protein that may impact antibody performance and subsequently raise reviewer concerns.

• Provide sufficient data or published support to show that the target protein is consistently expressed in your cell line or tissue

• Antibody reactivity can be affected by dynamic expression changes. Cell proteins can be modified, translocated, inserted into membranes, or even degraded

Target considerationsTarget Considerations Good PracticePre or pro protein Antibody epitope must be in pre/pro

region

Latent or activated proteinPhospho-specific or other PTM specific antibody

Tertiary structure obscures target Denaturing or degrading protocols needed

Complex native conformation or multi subunits

Conformation-specific antibody

Intracellular or intramembrane localization

Membrane disruption protocols needed

Live cell External cell surface epitope needed

Surface moiety Unfixed/light fixed frozen protocols or antigen retrieval needed

Reviewer comment: Can you submit data showing the antibody used only sees the trimethyl K27 site?

State-dependent antibodies must have extensive validation

Vendor should provide all validation data Protocols and control samples are often

available User control data is essential

Antibody design can impact assay performance

Polyclonals advantages and disadvantages

Advantages Disadvantages

Reviewer comment: Repeat your experiment with a monoclonal to be confident in your polyclonal data

Not a bad practice but not essential Choose a polyclonal made from short

peptide thus minimizing clonality Multi-application validation would help to

demonstrate specificity and robustness

Monoclonals advantages and disadvantages

Advantages Disadvantages

Reviewer comment: IHC staining seems inconsistent in figures throughout the manuscript

Lock down your protocol and order all antibodies from one lot

Confirm with monoclonal antibody Increase replicates Process slides in parallel

Target and tissue considerations

THE FIVE T’s!!!!!

Thickness?: slices/vibratome (>500um-50um); fixed frozen 40-15um; paraffin embedded (<10um- 0.5um) : accessibility of target; ability to wash out; resolution of final visualization.

The 5 “T”s of tissue prep for IHCTarget?: (epitope: protein, lipid, amino acid, chemical, integrin, growth factor, big, small, inside, outside, nuclear, mitochondrial, cytoplasmic?

Tissue? (expression of target? type of tissue? Brain, Liver, Skin, Cornea, Kidney, Bone?)

Time? : incubation times of: fixation, post-fix, Ab, washes, detection all make a difference and are interconnected. Fixes, influence penetration, epitope exposure, washing times, etc.

Temperature?: Kinetic energy of binding and interaction: hotter faster, colder slower –influences every step

Fixation shapes the epitope-antibody battleground

Light

Epitope distortion

Low

Fixation

Heavy High

TechniqueICC & Fresh frozen

Frozen sections

Paraffin

Plastic

Fixating on fixes: Procedures

Procedure Typical fix for ImmunohistochemistryIC (cells and cultures) 1-4% PFA, 50/50Ac/Meth coldSulfatides/glycoproteins (surface)e.g. O1, O4

No fix; Light PFA; no Ac/Me because they solubilize

IHC - frozen 50/50Ac/Meth, 2-4% PFAIntegrin adhesion molecules Bouins or light PFA fix; Perfusions Carnoy’s, 4% PFA, Bouin’s

IHC- whole mount 4% PFA, 10% Form, Carnoys

IHC - Paraffin 4% PFA, 10% Form, optional 2%Glut

IHC- EM 4-6% PFA w/ 2-4% Glut

• Dehydation and protein coagulation (air-drying; acetone, methanol, picric acid

• Formation of reversible cross-linkages (formalin, paraformaldehyde)

• Formation of strong, irreversible, fine scale cross-linkages (glutaraldehyde)

Will you be doing additional procedures? (PCR, ChIP, ISH/FISH, etc.)

The value of pre-experiment optimization and parallel processing

3 Cases:

• Titering• Parallel Controls• Multi-slide processing

The “5Ts” of tissue processing dramatically affect primary antibody concentration. Titering is critical despite the common practice of just using 1:1000 “unless the staining looks bad”

Antibody titering

EMD Millipore Antibody Optimization Project

OxyIHC™ labeled oxidative protein damagein the cerebellar cortex of an Alzheimer’sdisease transgenic APP mouse. Detection by anti-DNPH /HRP/DAB

Lapp, et al. 2010

Parallel controlsOptimizing tissue processing and key IHC steps together allows:

• Better controls• Assessment of signal/background • Staining comparisons• Identification of artifacts

Medium throughput multi-slide stainers are useful for these optimizations

WT control; no DNPH control APP hi exp; no DNPH control

WT control; DNPH+ DAB APP hi exp; DNPH+ DAB

Controlling variation among sample blocks and sections:Standardize tissue fixation/handling

NP-12676-3

NP-11761-32 NP-15395-3 NP-11714-1

NP-11572-1 NP-14181-1

Multi-slide processing

Yang, et al. 2015 Characterization of ERa phosphorylation sites as a marker for Tamoxifen resistant breast cancer, AACR

IHC of pER(Ser305) on ductal carcinoma

pER-S305 pER-S305

pER-S118ER

pER-Y537

pER-S106

pER-S167

pER-S102

Controlling variation among antibodies: Preoptimize each antibody used and then process in parallel.

Yang, et al. 2015, AACR

Multi-slide processing

Medium throughput Multi-slide stainers (e.g. SNAP i.d.® 2.0 system for IHC) can reduce sample variation

Single block IHC validation on ductal carcinoma tissue block NP14181-1

Some useful digital/analog resources

Humason’s Animal Tissue Techniques

Dako IHC Guidebook

EMD Millipore Introduction to Antibodies

Springer Practical IHC

IHCWorld

Histosearch

Histonet

Antibody design

Target and tissue

Pre-experiment optimization & parallel processing

In closing

Appendix 1: Fixatives in detail

56

In a fix: Acetone/Methanol50/50 Acetone/Methanol:

Acetone 100 ml

Methanol 100 ml

Mix well; use fresh at –20°C

• Uses- A light fix with minimal crosslinking- Fast penetration but gentle to cell structures- Excellent for cells and cultures

Light fix

In a fix: Carnoy’s

• Carnoy’s Fixative: glacial acetic acid 10 mLchloroform 30 mL100% EtOH 60 mL

• Uses- Made fresh, a light fix with minimal cross-linking- Fast penetration but gentle to cell structures- Excellent for light perfusion, frozen sections- Important for glycogen and Nissl body fixation- Good non-aldehyde fixation alternative- Chemically unreactive so minimal blocking of epitopes- Post-fixes in PFA give good long term preservation- Wonderful for integrins and adhesion molecules can be paraffin

embedded too for high resolution slices.

Light fix

In a fix: Bouin’s

Bouin’s Fluid:

Picric acid (saturated aqueous) 85 ml

Formalin (40% aqueous formaldehyde) 10 ml

Glacial Acetic Acid 5 ml

• Uses- A ‘hybrid’ fix with gentle crosslinking- Does not harden tissue- Good explosive when dehydrated

Light fix

In a fix: PLP - Aldehydes

Periodate-lysine-paraformaldehyde fixative

lysine-phosphate buffer 375mL

20% Formaldehyde 100mL

dH20 25mL

Sodium periodate 1.06g

• Uses- Mixed fresh, a moderate fix for cryostat sections - Excellent morphology but poor immunostaining.- Immunostaining greatly enhanced by brief prefix in acetone. - Paraffin embedding also maintains better antigenicity- Superior to acetone for fixation of membrane antigens, popular

in some hospitals; good for adhesion and epitope sensitive targets.

Moderate fix

In a fix: Aldehydes Buffered Formaldehyde (Formalin):

32.5g Na2HP04 and 20 g NaH2PO4 in water 4.5L

40% Formaldehyde 500mL

Adjust pH to 7.0-7.4

• Uses- A cheap strong fix with moderate crosslinking (particularly

between lysine residues)- Good but slow penetration but relatively gentle to protein

structure except when embedding.- Decent maintenance of antigenicity- Often used in archival and paraffin sections

Moderate fix

• Paraformaldehyde (PFA) 4%: Phosphate buffer 500mL granular PFA 20g >adjust to above pH12 to dissolve or use heat (toxic)

Adjust pH to 7.0-7.4 with NaOH MAKE FRESH; shelf-life 15 days

• Uses- Prepared fresh, an excellent easily made fix with moderate cross-linking (particularly between lysine residues)- Moderate penetration but gentle to protein structure- Faster fixation than formalin- Good maintenance of antigenicity- The closest to a ‘universal’ fix for Immuno and cell biology

In a fix: Aldehydes

Moderate fix

• Zenker’s stock solutionMercuric chloride 50g Potassium dichromate 25gSodium sulfate 10gDistilled water to 1 liter

Dilute 1:20 in glacial acetic acid for working solution

• Uses- Improves cytological preservation and minimize distortion associated with

formaldehyde-based fixatives (Often used as post fix to formalin)

- Coagulative crosslinking stabilizes many internal epitopes

- Good penetration of tissue but use sparingly as coagulative properties are

additive and can mask epitopes

In a fix: Mercuric Chloride based

heavy fix

• Paraformaldehyde (PFA) 4% & 2% Gluteraldehyde:

Phosphate buffer 500mL granular PFA 20ggluteraldeyde 10gAdjust pH to 7.0-7.4

• Uses- Prepared fresh, a fix with strong, irreversible cross-linking of many sites

on various macromolecules- Very fast fixation, moderate penetration- Deforms alpha helix structure in proteins so may significantly reduce

antigenicity- Gives excellent cytoplasmic and nuclear detail- Great for immuno of small molecules and Electron Microscopy

In a fix: Aldehydes

Heavier fix

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office To submit your

questions, click theAsk a Question

button

Webinar Series

Improving tissue-sample profilingThe optimization and application of immunohistochemistry

July 7, 2015

Sponsored by:

Nissi M. Varki, M.D.University of California San DiegoLa Jolla, CA

Kevin D. Long, Ph.D.EMD MilliporeTemecula, CA

For related information on this webinar topic, go to:

Look out for more webinars in the series at:

webinar.sciencemag.org

To provide feedback on this webinar, please e‐mailyour comments to webinar@aaas.org

Brought to you by the Science/AAAS Custom Publishing Office

Webinar Series

www.emdmillipore.com/snapihc

Improving tissue-sample profilingThe optimization and application of immunohistochemistry

July 7, 2015

Sponsored by: