ImmunostainingJuly 19, 2019

Alan K. Meeker, Ph.D.Departments of Pathology, Urology and OncologyJohns Hopkins University School of Medicine

Goal:Develop a basic understanding of tissue-based immunostaining, potential pitfalls, and how to avoid them

Outline:• Information obtained from immunostaining• Antibody basics• Tissue fixation and target retrieval• Basic methodology• Common problems• Finding good antibodies

Immunohistochemistry (IHC):utility in basic research

• Confirm genotype-phenotype (e.g. KO, KI, cell- or tissue-type specificity, etc.)

• Where is a biomarker expressed?• When is a biomarker expressed?• Co-expression or co-localization

(however, cannot prove direct physical molecular interaction)

• Cell type ID (via specific biomarkers) (e.g. CD4, CD8 T-cells, NK, Macrophages, Mast Cells;presence or absence; cell enumeration, etc.)

IHC: utility in translational research

• Discovery• ‘Omics Relevant in setting of interest??

• Pre-clinical• Animal models (e.g. is drug hitting the target?)

• Clinical• Dx, Differential Dx• Prognostic biomarker• Predictive biomarker• Monitoring patient status (Tx response or resistance)

IHC

• Localize specific molecules in tissues

• Based on antibody-antigen recognition

– Specificity– Sensitivity– Location!

Antibodies (immunoglobulins; Ig)

Target: typically a complex macromolecule (e.g. a protein)

Epitope: the portion of the target molecule that the antibody recognizes and binds to.

Specificity and Affinity for the target vary between diff. Abs

Enormous diversity in recog.!

Epitope

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 1 of 11

Antibodies

Primary Ab

Secondary Ab

Antigen-antibody complex

Antibody characteristics

Polyclonal – a mixture of multiple different antibodies recognizing different epitopes of the same antigen.

Monoclonal – identical copies of the same unique antibody. Recognizes only a single epitope.

SensitivitySpecificity

Specimens• Cells

– From organs/tissues (FNA, smears, touch imprints)– Cultured cells

• Tissues– Frozen sections – “Fixed” (usu. formaldehyde), processed & embedded

in paraffin (wax) blocks • Formalin-Fixed Paraffin Embedded = “FFPE”

FixationGoals

1. Prevent self-digestion (autolysis) by rapidly terminating enzymatic/metabolic activities

3. Prevent bacterial decomposition.

2. Preserve tissue structures while stabilizing and hardening the tissue for processing.

FixationThe Ideal Fixative

1. Acts rapidly.

2. Safe/easy to use.

3. Inexpensive.

4. Stabilizes tissue, allows for handling and processing.

5. Preserves cellular and tissue structures without introducing artifacts.

DOES NOT EXIST

Tissue Fixation

GARBAGE IN – GARBAGE OUT

Originally optimized for preserving morphology and for histochemical staining:NOT for detecting antigens with antibodies!

Proper fixation is critical to the success of downstream immunostaining procedures!

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 2 of 11

• Formaldehyde

– Gas, soluble in water.

– “Formalin” = 37% formaldehyde solution10% neutral buffered formalin “NBF” = 4% formaldehyde + stabilizers.

- Reacts primarily with amino groups in proteins forming covalent cross-links

1. Fixative type and Concentration2. Salts (osmolality)3. pH4. Temperature5. Incubation Time6. Tissue type

Key factors affecting chemical fixation

LACK OF STANDARDIZATION: EACH LAB’S TISSUES UNIQUE!!

F

Formaldehyde

• Specimen size is important!

• Diffusion time

• Reaction time

Formalin

Fixation gradient

Anti-CD45 IHC in lymph node

https://www.slideshare.net/appyakshay/immunohistochemistry-75139545

Formalin: minimum of 15-20 tissue volume equivalents!

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 3 of 11

Perfusion Fixation

From: Atlas of Plant and Animal Histologyhttps://mmegias.webs.uvigo.es/02-english/6-tecnicas/2-metodos-fijacion.php

Fix tissues

Remove water with alcohol (ethanol; dehydration)

Remove ethanol and fats with organic solvent (xylene or toluene; “clearing”)

Infiltrate tissues with melted paraffin wax (~60 degC)

Embed the tissues in a paraffin block

Tissue processing

70% 100%

Paraffin block

Fixation crosslinks can block antibody access to target epitopes.

Ag

nucleus

cytoplasm

Treatment with protease can re-exposeepitopes! (“antigen retrieval”)

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 4 of 11

Heat Induced Epitope Retrieval (HIER)

• Reverses cross-links• Aids in rehydration, protein renaturation• Overall, heat appears to be the key factor

Optimal retrieval method is determined empirically.- tissue type- details of fixation, storage- antibody/target

Key variables

Lab-to-lab variability!

A variety of different retrieval buffers and heat sources (e.g. microwave, steamer, autoclave, etc.)

No pretreatment

1N HCl pretreatment

Citrate pH=6 heat pretreatment

Citrate heat + 1N HCl pretreatment

Pretreatment example: 5-OH-methyl-C antibody (DNA modification)

Immunostaining

Detection of the Ab-Ag complex:

Direct vs. Indirect

Immunofluorescence

A.H. Coons 1941 direct labeling of antibody with fluorescent dye on frozen sections

http://en.wikipedia.org/wiki/Image:Immunohistochemicalstaining1.PNG

Direct version

http://en.wikipedia.org/wiki/Image:Immunohistochemicalstaining2.PNG

Indirect method (typically used today)

Primary Ab

Secondary Ab

Immunofluorescence

In Vitrogen/Molecular Probes

A wide variety of fluorescent dyes

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 5 of 11

Immunofluorescence• Advantages:

– Hi-resolution– Multiplexing (use of

multiple different Ab) – Better subcellular detail– Can be used with 3D

microscopy/live imaging

• Disadvantages: – Tissue background

(autofluorescence)– Cost (equipment) – Less surrounding tissue

detail – Impermanent

Actin (blue) Mitochondria (red)Histones (green)

Immunohistochemisry (IHC)• Antibodies linked to enzymes

-generates an intensely colored productvisible with conventional microscopy

Y

Colorlesssubstrate

Colored end-product

E

http://en.wikipedia.org/wiki/Image:Immunohistochemicalstaining2.PNG(modified)

Indirect method

Primary Ab

Secondary Ab

Chromogenic IHC

Enzyme (e.g. HRP) **enzymesubstrate

* *****

Anti-Alpha-methyl CoA Racemase (prostate)

Cancer Pre-cancer

Normal

Multiplexing with IHC

(Dako “Envision+” kit)

Dextran polymer technology= HRP enzyme

Signal Amplification

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 6 of 11

1:200 Ab dilutionLeica Power Vision+ Kit

1:50 Ab dilutionDAKO Envision+ Kit

Anti-p16 Signal amplificationExample (IHC)

Immunofluorescence Signal Amplification

Y

Catalyzed signal amplification (CSA)

TonsilCD4 + Foxp3

“Reproducibility crisis” in biomedical res.

• Human Protein Atlas tested 5,000 Commercial Ab. <50% worked for IHC in fixed tissues.

• Kinexus Bioinformatics tested 6,000. >3/4 non-specific or non-functional

350 companies>2 Million Ab for research$2 Billion global market$350 Million est. losses to U.S.

• Largely avoidable with use of proper controls!!!

Batch-to-batch antibody variabilityEsp. polyclonalsCheck with vendor to confirm same lot

Off-target artifacts (Ab specificity prob.)Worst case: Ab not even recog. target!False Neg.False Pos.Check data on specificityValidate in-house with rigorous controls!

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 7 of 11

Case study 1: telomerase (hTERT)

TMA-7 CSA II

Bio SB“anti-hTERT”Monoclonalantibody

PitfallsPPiittffaallllss

Journal of Cell Science 119: 2797-2806, 2006.

Ab validation using cell line controlsA

ntib

ody

YA

ntib

ody

XN prostate

(+Telomerase)N prostate N breast Mouse telomerase

KO fibroblasts

Case study 2: mouse GSTP1

GSTP1 Ab (1:10,000)

Contributed by Dr. Angelo De Marzo

Wild type mouse

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 8 of 11

Contributed by Dr. Angelo De Marzo

GSTP1 Ab (1:10,000)

Knock out mouse(GSTP1-/- )

Finding antibodies:Useful information sources

1. The publishedliterature

2. Antibody Data Sheets

Western Blotting gives info onSpecificity ?

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 9 of 11

Keep your eyes open!

3. Web resources

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 10 of 11

4. ColleaguesSummary

• Immunostaining is a powerful, informative technique• Many basic/translational research applications• A variety of detection strategies

• Direct/Indirect• Fluorescent/Chromogenic• Multiplex staining options

• Several variables can impact IHC results• Tissue type• Fixation• Pre-treatment (heat, protease…)• Ab selection (species, class, monoclonal,

polyclonal…)

Summary• Antibody validation is critical (if not already done)

• Do your homework first!• Utilize available resources• Saves time, money and grief!• The need for appropriate (+/-) controls cannot be

over-emphasized!!!

Summary

2019 JH Phenocourse Meeker IHC

Educational Use Only Page 11 of 11