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Immunostaining 101
July 19, 2013
Disclosures Goal
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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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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
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 *
Immunostaining Immunofluorescence
Direct method
1N HCl pretreatment
31 Citrate heat + 1N HCl pretreatment 32 33
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 Meeker - Immunohistochemistry - P 5
• Advantages: Immunohistochemisry (IHC) • Peroxidase (HRP) – enzyme w/ high
– Hi-resolution, easy to
turnover rate producing good sensitivity
double/triple label • Antibodies cross-linked to enzymes that – Most commonly used
– Better subcellular detail – Most often used substrate:
generate an intensely colored reaction end
– Can be used with 3D 3,3'-Diaminobenzidine Tetrahydrochloride (DAB)
microscopy/live imaginjg product visible with conventional bright
(rapid oxidation and polymerization of DAB by H202)
Actin (blue) field light microscopy
Mitochondria (red)
• Disadvantages: Histones (green)
Colorless
– Background substrate
autofluorescence Colored end-product
– Cost
E
– Lack of surrounding
tissue/cellular detail
Y
– Not permanent
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IHC example: p27 in prostate luminal cells (HRP/DAB) 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 Basal • VECTOR SG
• VECTOR VIP = PURPLE
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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)
1:200 Ab dilution
Y
Power Vision+ Kit
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Information sources
The published
literature
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Antibody
Spec
Sheets
Check out
the images!!!
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• Colleagues
• Sales reps – FREE samples! • Largely avoided with use of proper controls!!!
Pitfalls
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”)
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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
Embed
PBS
10%
formalin-
buffered
phosphate
Negative Ab – Ab –
H&E
control high dilution low dilution
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Negative control
(Primary antibody omitted)
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
• Antibody work-up
• Do your homework first!!!!
• Use of appropriate +/- controls cannot be over-emphasized!!!
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