Meeker - Immunohistochemistry - P 1

Immunostaining 101
July 19, 2013
Disclosures Goal

No relevant financial interests to disclose – Gain an understanding of the basic

concepts and practical aspects of
immunostaining

Alan K. Meeker, Ph.D.

Departments of Pathology, Urology and Oncology
Johns Hopkins University School of Medicine

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Immunostaining Antibodies (immunoglobulins; Ig)

WHY do immunostaining?
• Localize specific
molecules in tissues Epitope Enormous diversity!
• confirm genotype-phenotype
• based on antibody- Target Ag: typically a small portion of a
(e.g. KO, KI, levels or efficiency of expression, etc.) complex macromolecule (e.g. a protein)
antigen recognition
• where is a biomarker expressed? – proteins, phospho- Specificity and Affinity for Ag can vary
• when is a biomarker expressed? peptides, adducts,
sugars, lipids, etc
• co-expression or co-localization.
• cell type-specific biomarkers – Specificity
(e.g. CD4, CD8 T-cells, NK, Macrophages, Mast Cells, etc.)
– Sensitivity

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Antibodies Antibody characteristics Antibody characteristics

SPECIES OF ORIGIN - mouse, rat, rabbit, chicken, goat, sheep, etc.
Polyclonal – a mixture of multiple antibodies recognizing different
Secondary Ab CLASS - Important detail for selection of appropriate secondary Ab epitopes of the antigen.

CHICKEN!

Primary Ab (IgY)

Antigen-antibody complex
Fc Monoclonal – identical copies of the same unique antibody.
Recognizes only a single epitope.

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 Meeker - Immunohistochemistry - P 2
Antibody characteristics Specimens Fixation
Polyclonal • Cells Goals of fixation
1. in general produce stronger signals
– From organs/tissues (FNA, smears, touch imprints)
2. greater potential for false positive staining due to 1. Prevent autolysis by rapidly terminating
antibodies cross-reacting to undesired targets – Established cell lines (coverslips, chamber slides, enzymatic/metabolic activities
(affinity purification helps) cytospins, cells embedded in paraffin, etc.)
3. limited supply 2. Preserve tissue structures while stabilizing
• Tissues and hardening the tissue for processing.
Monoclonal
1. highly specific – Frozen sections 3. Prevent bacterial decomposition.
2. less background – “Fixed” (e.g. formalin-fixed) paraffin embedded blocks
3. intrinsic cross-reactivity to non-target can be problematic
4. potential for epitope loss = loss of staining
5. unlimited supply

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Tissue Fixation Tissue Fixation Fixatives

-Aldehydes (formaldehyde, glutaraldehyde).
The Ideal Fixative DOES NOT EXIST
Optimized for maintaining -Oxidizing agents, metallic ions and complexes
1. Acts rapidly. morphology/histochemical staining, NOT (osmium tetroxide, chromic acid, Zn).
for detecting antigens with antibodies! -Protein-denaturing agents
2. Safe/easy to use. (acetic acid, methanol, ethanol).

3. Inexpensive. Proper fixation is often critical to -Poorly-understood mechanisms

(mercuric chloride, picric acid).
4. Stabilizes tissue, allows for handling and the success of downstream
-Other: microwave, vapor fixation.
processing. immunostaining procedures!!!
5. Preserves cellular and tissue structures -Combinations of above.
without introducing artifacts.
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Key factors affecting chemical fixation

 Formaldehyde  Formaldehyde
1. Temperature
2. Osmolality
– Gas, soluble in water. – Takes time to penetrate,
3. pH
THEN rate of fixation is relatively slow
4. Concentration of fixative
– “Formalin” = 37% formaldehyde solution
5. Incubation time
6. Tissue type - 10% buffered formalin (“NBF”)
= 4%formaldehyde+stabilizers.
* 7. Size of specimen !
(fixative penetration) - Reacts primarily with free amino groups
in proteins to form covalent cross-links
(Lys, Arg, Asp).
LACK OF STANDARDIZATION - EACH LAB’S TISSUES UNIQUE!!
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 Tissue processing Meeker - Immunohistochemistry - P 3
Paraffin block
Fix tissues (~ 2mm thickness) in 15 Vol. 10% buffered
formalin at room temp; usu. overnight.
70% 100%
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

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Orientation
cloverleaf

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22 23 JHU Tissue Microarray ~ 400 cores, 0.6 mm ea. 24 JHU Tissue Microarray – H&E stained

Retrieving antigens “lost” during fixation  Formaldehyde How to retrieve antigens “lost” during fixation

ƒ Frozen sections were commonly used to – Cross links can block antibody access to • Protease digestion
bypass the problem of ”over fixation” target epitopes.
– Treatment with protease can re-expose • Several types
but cumbersome and gives poor morphology -trypsin, chymotrypsin, papain,
epitopes! (“antigen retrieval”)
protease VIII, etc., freshly prepared
• Limited protease treatment could allow • Key Parameters:
successful antibody staining in previously
• Enzyme
negative tissues
• Temperature
• Buffer (pH)
Thus: epitopes were not really lost!! • Enzyme concentration
• Incubation time *

Ag *usually hold other parameters constant and vary this

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 The “Hot Revolution!” HIER HIERMeeker - Immunohistochemistry - P 4
• Early 1990s: two groups published that heat treatment
could dramatically improve immunostaining results
Pros: Overall, heat appears to be the key factor.
*HIER, - Improved signals - reverses cross links
• HTTR, AR, etc.
- Less background - aids in rehydration
• A variety of buffers - protein renaturation
• citrate pH 6.0 - Previously unusable antibodies now work!!
Cons: - calcium chelation
• low pH
• high pH - Increased biotin (background)
• EDTA pH 8.0 (Ca++ removal?) Optimal method is determined empirically.
- Some false pos./false neg.
• commercial formulas - tissue type
- Loss of tissue integrity (esp. fibrous and
fatty tissues) - details of fixation Key variables
• A variety of heat sources
• microwave - Results may conflict with older literature - antibody/target
• heat plate (immersion)
• steamer
• pressure cooker/autoclave UNFORTUNATE lab-to-lab variability!
*HIER = Heat Induced Epitope Retrieval
HTTR = High Temp Target Retrieval
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Pretreatment example: 5-OH-methylC antibody

Immunostaining Immunofluorescence
Direct method

Detection of the Ab-Ag complex:

No pretreatment Citrate heat pretreatment • A.H. Coons in 1941
Direct vs. Indirect with direct labeling of
antibody with
fluorescent dye,
worked on frozen
sections http://en.wikipedia.org/wiki/Image:Immunohistochemicalstaining1.PNG

1N HCl pretreatment
31 Citrate heat + 1N HCl pretreatment 32 33

A wide variety of fluorescent dyes

Immunofluorescence Indirect method advantages:
Indirect method -versatility and convenience
- increased sensitivity

Polyclonal
Secondary Ab Secondary Ab

Primary Ab Primary Ab

In Vitrogen/Molecular Probes
34 http://en.wikipedia.org/wiki/Image:Immunohistochemicalstaining2.PNG 35 http://en.wikipedia.org/wiki/Image:Immunohistochemicalstaining2.PNG 36
 Immunofluorescence
• Advantages:
– Hi-resolution, easy to
double/triple label
– Better subcellular detail
– Can be used with 3D
microscopy/live imaginjg
• Disadvantages:
– Background
autofluorescence
– Cost
– Lack of surrounding
tissue/cellular detail
– Not permanent
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IHC example: p27 in prostate luminal cells (HRP/DAB)
Basal
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Other Enzymes
• Alkaline phosphatase
– 5-bromo-4-chloro-3-indolyl phosphate/nitroblue
tetrazolium (NBT)
– Vector Red
– Vector Blue
– Vector Black
– etc
• Glucose oxidase
– with nitroblue tetrazolium
• B-D-Galactosidase
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Meeker - Immunohistochemistry - P 5
Immunohistochemisry (IHC) • Peroxidase (HRP) – enzyme w/ high
turnover rate producing good sensitivity
• Antibodies cross-linked to enzymes that – Most commonly used
– Most often used substrate:
generate an intensely colored reaction end
3,3'-Diaminobenzidine Tetrahydrochloride (DAB)
product visible with conventional bright
(rapid oxidation and polymerization of DAB by H202)
Actin (blue) field light microscopy
Mitochondria (red)
Histones (green)
Colorless
substrate
Colored end-product
E
Y
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Alpha-methyl CoA Racemase in prostate
Other Peroxidase Substrates
Ca
• 4-chloro-1-naphthol = BLUE
• 4-napthol pyronin = RED-PURPLE
• 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS)
• 3-amino-9-ethylcarbazole (AEC) = RED
• VECTOR Nova = RED
Basal • VECTOR SG
• VECTOR VIP = PURPLE
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Multiplex immunolabeling 3 Colors, 4 Antibodies
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T. Iwata, AM De Marzo, unpublished
 Indirect IHC Methods w/Amplification Indirect Methods w/Amplification Meeker - Immunohistochemistry - P 6
• Direct labeling: cumbersome and not very sensitive
Multiple HRP enzymes
(although newer kits available make it easier)
• Indirect methods now most common: Dextran polymer technology
Y Secondary antibody
Avidin Biotin Complex = HRP enzyme
= Biotin

= HRP enzyme
Y Primary antibody
Biotinylated-HRP
= Avidin

1. Biotinylated secondary
“PowerVision+” IHC Detection System (Leica)
2. ABC complex binds to secondary
3. Enzyme in ABC converts substrate (e.g. DAB)

Ventana Chem-Mate (e.g. Dako “Envision+”)

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Example of signal amplification

Anti-p16
Catalyzed Signal Amplification Immunofluorescence with
Catalyzed signal amplification (CSA)
1:50 Ab dilution • CSA kit (DAKO) – Enzymatically deposits many biotin
DAKO Envision+ Kit molecules using HRP and biotinyl tyramide
-Detected with streptavidin-HRP

• CSA kit II (DAKO) – biotin-free signal amplification

1:200 Ab dilution
Y
Power Vision+ Kit
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Blocking (reduces background) Standard workup of antibodies for immunostaining

Practical aspects of
immunostaining Sources of background staining:
• Endogenous enzymes
-Peroxidases*
Peroxidase block with H2O2
-Alkaline Phosphatases
Levamisole
• Endogenous biotin**
Avidin, biotin block
• Non-specific Ab binding sites
Protein block (BSA, etc.)
Main process variables
1. Antigen retrieval
– Citrate, HTTR, microwave, pressure cooker, etc.
Blocking is performed 2. Primary antibody dilution
BEFORE adding the – Perform a dilution series* of primary antibody
primary antibody
3. Detection system
– Direct, Indirect, polymer detection, etc.

• *Red blood cells, Leukocytes, Spleen, Bone Marrow

• **Liver, Kidney, Lung, Spleen, Brain, Adipose, Mammary gland *1 ug/ml Ab is good starting point if no other info is available
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 Meeker - Immunohistochemistry - P 7

What if doesn’t work??! (low or no signal)…

– Try greater amplification (CSA kit, etc.)
– Incubate the primary overnight at 4 degrees C
– Try high pH or EDTA pre-treatment solution
– Try protease digestion (usu. avoid unless recommended)

– Obtain additional antibodies!! $$$

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Information sources
The published
literature

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Antibody
Spec
Sheets
Check out
the images!!!

Immunohistochemistry of the ezrin protein in lung

adenocarcinoma. Unlike radixin or moesin, weak to
moderate staining for ezrin was seen in most tumor cells (A,
C, and D), except for one tumor which was completely
negative (B). Localization of ezrin was altered in a
significant proportion of tumor cells. Whereas tumor cells
forming luminal structures retained membranous staining
on the apical side (A), the majority of tumor cells were
diffusely stained in the cytoplasm (C). This was especially
evident in tumor cells with disorganized structures. In
addition, cancer cells invading the fibrous stroma in a
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scattered manner were strongly stained for ezrin (D). A to 62 63
D, Original magnification, 100; E, higher magnification of D
(original magnification, 250).
 Meeker - Immunohistochemistry - P 8

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Problems & Pitfalls

Bio SB
“anti-hTERT”
Monoclonal
• Other web sites antibody

• Colleagues

• Sales reps – FREE samples! • Largely avoided with use of proper controls!!!

– Tissue samples known to express/not express the

antigen of interest.

– KO cells (-) versus same cells expressing

epitope-tagged gene (+++)
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TMA-7 CSA II

Pitfalls

Journal of Cell Science 119, 2797-2806, 2006

WT mouse GSTP1-/-
GSTP1 Ab (1:10,000) GSTP1 Ab (1:10,000)

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Contributed by Dr. Angelo De Marzo Contributed by Dr. Angelo De Marzo
 Potential Problems & Pitfalls Meeker - Immunohistochemistry - P 9
Use of Cell Line Controls MYC Western
Example: MYC Staining with RNAi Knockdown
• Enzyme detection methods:

CWR22rv1
P493 +Tet
P493 -Tet

LNCaP
– Non-specific antibody binding (example: tumors can be “sticky”)

Molecular Weight (kDa)

– Cross reactivity with another antigen - can be very difficult to solve!
(e.g. DNMT1, GSTP1, hTERT antibody). 115

– Endogenous enzyme activity (e.g. block peroxidase with H2O2) C-MYC 82

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– Endogenous biotin (if biotin based system; block with avidin+biotin 47
block) 39
– Endogenous antibody-binding activity (Fc) (block with protein block
or normal serum)
– Endogenous antibody (“mouse on mouse” – use of mouse 82
monoclonal on mouse tissue) 64
ACTIN 47
– Retrieval Conditions (protease vs. steam can make-or-break)
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Transfected with Scrambled siRNA Transfected with MYC-targeted siRNA

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C. Koh, Unpublished

C-MYC Staining in Human Prostate Tissues Making control cell line pellets Fixed cell line staining controls

Step 1

Snip off cap

2% agarose (in PBS)

cells

Embed
PBS

10%
formalin-
buffered
phosphate

Negative Ab – Ab –
H&E
control high dilution low dilution
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B. Gurel, T. Iwata, C. Koh, et al, AM De Marzo, Modern Path, 2008 In Press

Pretreatment: HTTR Pretreatment: HTTR Pretreatment: HTTR

Optimizing Antibody Staining Ab dilution: 1:250 Optimizing Antibody Staining Ab dilution: 1:2000 Optimizing Antibody Staining Ab dilution: 1:1000
NQ01 Detection: HRP+DAB NQ01 Detection: HRP+DAB NQ01 Detection: HRP+DAB

Treated Untreated Treated Untreated Treated Untreated

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 Meeker - Immunohistochemistry - P 10

Negative control
(Primary antibody omitted)

Pretreatment: HTTR Pretreatment: HTTR

Ab dilution: 1:1000 No primary antibody
Detection: HRP+DAB Detection: HRP+DAB
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Anti-CD4 antibody on mouse spleen Anti-CD4 NO PRIMARY ANTIBODY on mouse spleen Anti-CD4 antibody on mouse spleen
(Quanto; Thermo Sci.) “Mouse on Mouse” kit

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Summary
• Immunostaining is a powerful, informative technique
• Localization of a molecular moiety by use of an antibody
• A variety of detection strategies
• Direct/Indirect
• Fluorescent/Chromogenic
• Sensitivity/Specificity

• Several variables to consider

• Tissue type
• Fixation
• Pre-treatment (heat, protease…)
• Ab selection (species, class, monoclonal, polyclonal…)

• Antibody work-up
• Do your homework first!!!!
• Use of appropriate +/- controls cannot be over-emphasized!!!

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