2019 Annual Tri-State Plus One Histology Symposium Strategies To Identify and Eliminate Background Interference in IHC and IF Tissue Section Applications Craig Pow, Ph.D. Director, Technical Service [email protected]16 th Annual Tri-State Plus One Histology Symposium Des Moines, IA May 8-10, 2019
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Strategies To Identify and Interference in IHC and IF Tissue · Key Steps in Two-Step (Strept)Avidin/Biotin IHC Detection Procedure Staining with Second Deletion Control Avidin/Biotin
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2019 Annual Tri-State Plus One Histology Symposium
Strategies To Identify and Eliminate Background Interference in IHC and IF Tissue Section Applications
2019 Annual Tri-State Plus One Histology Symposium
Identify the Source(s) of the Background
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Run Defined Controls
▪ Positive (Protocol Optimization)
▪ Negative (Deletions)
Primary Antibody Incubation Enzyme Polymer Incubation React with Substrate
Key Steps in One-Step Polymer IHC Detection Procedure / Workflow
2019 Annual Tri-State Plus One Histology Symposium
But …..
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“..nothing has changed.”
2019 Annual Tri-State Plus One Histology Symposium
Well something has …
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1.
2.
3.
Times, Temperature, Washes
Antigen RetrievalProtocol Optimization
(Positive Control)
Heterogenous Tissue Block
▪ Cell Type
▪ Antigen Expression
▪ Vascularization
▪ Inflammation
▪ Necrosis
2019 Annual Tri-State Plus One Histology Symposium
First Deletion Negative Control
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Apply Substrate Working Solution Only (Omit Other Detection Reagents)
▪ Treat section(s) as per SOP.
▪ Just prior to applying primary antibody, add substrate working solution.
▪ Apply substrate for “pre-determined” time.
▪ Do not counterstain (i.e. no hematoxylin).
▪ View.
Determines Effectiveness of Quenching Step Reagents(Endogenous Enzyme Activity)
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Issues arising from:
▪ Lack of quenching (omitted; diluted/old reagent; time)
▪ Inappropriate enzyme (HRP or AP) quench
Staining with First Deletion Control
Endogenous alkaline phosphatase (AP) and peroxidase (HRP) activities in frozen, acetone-fixed intestine section revealed with AP substrate (magenta) and HRP substrate(brown). Hematoxylin counterstain (blue).
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WITHOUT WITH
Quenching Endogenous Enzyme Activity in Tissue Sections
Presence of endogenous peroxidase activity indicated by application of substrate (DAB) alone, no other detection reagents.
▪ Closely related species➢ Anti-mouse IgG on rat tissue (vice versa)
✓ Use pre-adsorbed reagent✓ Dilute in serum from specimen
Without Specific Mouse Ig Block
With Specific Mouse Ig Block
Staining with Second Deletion Control
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Blocking:
▪ Verify appropriate serum (dilution, time)▪ Alternatives to normal sera (e.g. gelatin or animal free)▪ Add detergent to wash buffer (0.1% - 0.5%)
WITHOUT Animal-Free Block WITH Animal-Free Block
Adjacent tissue sections stained with HRP/DAB method without protein block and with protein block (sera alternative animal-free block). No counterstain.
Staining with Second Deletion Control
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▪ Inadequate buffer washes
▪ Concentration too high
▪ Decrease incubation time
Protocol OptimizationCheck with Positive Control Data
Adjacent sections exposed to varying buffer wash times after polymer incubation step.Hematoxylin Counterstain.
Shorter Wash Time Longer Wash Time
Staining with Second Deletion Control
Other Contributing Factors:
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Primary Antibody Incubation
Biotinylated Secondary Incubation
Enzyme conjugated (Strept)Avidin / ABC
React with Substrate
Key Steps in Two-Step (Strept)Avidin/Biotin IHC Detection Procedure
Staining with Second Deletion Control
Avidin/Biotin (non-polymer) based Deletion Controls
➢ 0.05% avidin + 0.005% biotin in buffer for effective blocking▪ Substrate & (Strept)Avidin enzyme Conjugate & Biotinylated Secondary
2019 Annual Tri-State Plus One Histology Symposium
Complete Detection System
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Inappropriate Staining
▪ Ensure primary antibody has been validated (commercial source / purified?)➢ Recommended guidelines:
✓ Titer✓ Time✓ Temperature✓ Diluent
▪ Non-purified primary antibody (e.g. ascites, culture media, whole sera)➢ Run titer series➢ Dilute in buffer with blocking agent (e.g. serum and/or detergent)➢ Incubate in buffer containing 2% - 5% normal serum derived from the
same species as the tissue. ~ 1 hr at R/Temp prior to applying to tissue.
Check positive control tissue
2019 Annual Tri-State Plus One Histology Symposium
Appropriate Negative Controls:
1) Substituting primary antibody for a non-immune Ig.
2) Preadsorption of the primary antibody with the immunogen used to generate the primary antibody.
3) Use an irrelevant primary antibody.
4) Removal of target antigen (digestion). Treated vs Untreated.
What Is An Appropriate Negative Control?
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Simply omitting the primary antibody
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Further Tools and Resources
Resource Tools
IHC Literature Texts / JournalsWorkflowsProduct Selection Guides Troubleshooting Guides
Vendor Websites ProtocolsData SheetsTechnical Support
Video Tutorials JOVEYouTube
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Familiar with IHC Workflow
Positive Controls
Negative Controls
Troubleshooting
Implement Adjustments
Clear, Unambiguous View of Target Antigen
Summary
GOAL:
2019 Annual Tri-State Plus One Histology Symposium
Questions?
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Part 2: Immunofluorescence
▪ Introduction to fluorescence
▪ Introduction to immunofluorescence
▪ Sources of background fluorescence (autofluorescence) in immunofluorescence
▪ Conventional solutions to reduce autofluorescence
▪ New solutions to reduce autofluorescence
Introduction
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Fluorescence
Definition: Absorption of electromagnetic radiation at one wavelength and re-emission at another lower energy wavelength.
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Immunofluorescence
▪ Determination of the location of an antigen using an antibody labelled with a fluorescent dye (fluorophore) (direct)
▪ Usually, a primary antibody that is not labelled is used to bind the antigen, and a secondary antibody follows that is labelled with a fluorophore (indirect)
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Fluorescence Microscopy
▪ Standard (epifluorescence) vs. laser scanning confocal microscopy
▪ Light sources (LED, metal halide, mercury, xenon, laser) have different spectral distributions. Some will illuminate brighter in the UV region than others for example.
2019 Annual Tri-State Plus One Histology Symposium
Sources of Background Fluorescence
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Arise from one or more sources:▪ Inherent to the specimen – “Autofluorescence”▪ Detection reagents – e.g. Primary / Secondary Antibodies▪ Combination of tissue & reagents
Unwanted non-specific fluorescence observed on the test specimen.
Fluorescent compounds endogenous and/or introduced on the specimen
Don’t be like this guy.
Get a clue and identify the background source(s)
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Basic IF Workflow
Untreated
Sodium borohydride Sudan Black B
Step 1: Tissue Preparation (Antigen Retrieval)
Step 2: Protein Blocking
Step 3: Primary Antibody Incubation
Step 4: Secondary Antibody Fluorophore Conjugated
Step 5: Coverslip with Antifade Medium
Step 6: Visualize
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Chemical Treatments
▪ Can be destructive to tissue (KMnO4, H2O2)
▪ Can be inconvenient and not reproducible (NaBH4)
▪ Generally not very effective for most autofluorescence
▪ Most autofluorescence is due to cross-links in tissue, and these are difficult to break once fixed and mounted
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Photo Treatments
UV or Visible Light Exposure:
▪ Illuminating with high intensity light to irreversibly photo-oxidize fluorescent elements
▪ Reasonably effective for some tissue types, however requires 24-48 hours of treatment, and requires lamps and equipment that many laboratories do not possess
▪ Not convenient for large numbers of slides
▪ Not effective for lipofuscin
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Masking Treatments
A hydrophobic dye molecule binds to the tissue and absorbs (masks) incident radiation (also called dark quenching)
▪ Sudan Black: good for lipofuscin; introduces fluorescence in far-red
▪ Eriochrome Black T: somewhat effective for FFPE tissue
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Quenching Treatments
Copper Sulfate
▪ Can be used to quench the fluorescence of the tissue. Copper in the +2 oxidation state can readily accept electrons from excited state molecules and quench them.
▪ However the same mechanism quenches the fluorophore on the secondary if copper is remaining in the sample.
▪ When tissue is washed with buffer, most of the copper washes away.
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Previous Investigations
Untreated Eriochrome Black T
Sodium Borohydride Sudan Black B
FFPE Human Tracheal Tissue.
Reference:Davis, et al., (2014) J. Histochem Cytochem, 62(6):405-423
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Previous Investigations
Yang J, Yang F, Campos LS et al. Quenching autofluorescence in tissue immunofluorescence. Wellcome Open Res 2017, 2:79 (doi: 10.12688/wellcomeopenres.12251.1)
▪ Ultraviolet (UV)▪ Ammonia (NH3)▪ Copper (II) sulfate (CuSO4)▪ Trypan Blue (TB), ▪ Sudan Black B (SB), ▪ Commercial Ink Based Reagent
& combinations of these treatments could reduce AF in paraffin and frozen sections of placenta and teratoma in FITC, Texas Red and Cy5.5 channels.
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Latest Advances
Untreated
Sodium borohydride Sudan Black B
Most Recent Technology:
▪ A solution based on hydrophilic molecules binding to tissue that combines the effects of masking and quenching.
TrueVIEW Autofluorescence Quenching Reagent:
▪ Aqueous, Non-Fluorescent
▪ Short Treatment (2-5 minutes)
▪ Short Wash After Treatment (2-5 minutes)
▪ Can Be Scaled Up For Increased Slide Volume
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Comparison of Autofluorescence Reduction (AFR) Treatments
Untreated
Sodium borohydride
Copper Sulfate Sudan Black B Sodium Borohydride
TrueVIEW
Untreated Human Pancreas
Red blood cellsFormalin fixation
Collagen
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Mode of TrueVIEW Treatment
Untreated
Sodium borohydride Sudan Black B
Specific signal (fluorophore labelled secondary antibody)
Fluorescent endogenous “elements”
AFR Treatment
Specific signal retained, with background signal reduced
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Human Spleen
Sodium borohydride
Autofluorescence in green channel
Autofluorescence in red channel
TrueVIEW Treatment
TrueVIEW Treatment
Adjacent human spleen tissue sections double stained using primary antibodiesagainst CD20 (red) and Ki67 (green) antigens. Left, untreated; right treated. White arrows indicated true antigen staining.
Without Treatment With AFR Treatment
2019 Annual Tri-State Plus One Histology Symposium
If You Encounter AF …
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2019 Annual Tri-State Plus One Histology Symposium
…Glove Up and Pinpoint the Source
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Tissue elements and/or use of aldehyde fixative
Lipofuscin
TrueVIEW AFR(Vector)
MaxBlock AFR Kit (MaxVision)
FluoMute AF Blocking Agent(Neuromics)
AutoFlu. Eliminator Reagent(Millipore/Sigma)
TrueBlack (Biotium)
AF Source Treatment
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Conclusions
Sodium borohydride
Autofluorescence in red channel
2) Determine the source of the AF – lipofuscin, tissue elements, aldehyde induced.
3) Once the source has been identified, apply appropriate measures to reduce the AF.
1) Beyond the detection reagents, fixation and endogenous tissue elements can introduce significant autofluorescence (AF) background in an immunofluorescence (IF) assay.
4) Run assay again and ensure AF has been reduced in the part(s) of the spectrum you are targeting.
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Sodium borohydride
Autofluorescence in green channel
Autofluorescence in red channel
TrueVIEW Treatment
TrueVIEW Treatment
Questions?
2019 Annual Tri-State Plus One Histology Symposium