Strategies To Identify and Eliminate Background Interference in IHC and IF Tissue Section Applications
Craig Pow, Ph.D. Director, Technical Service [email protected]
16th Annual Tri-State Plus One Histology Symposium Des Moines, IA May 8-10, 2019
2019 Annual Tri-State Plus One Histology Symposium Objectives
Upon completion of this workshop, participants will be responsible to: ▪ Identify Sources of Non-Specific Staining in Tissue Based Assays
▪ Evaluate Options Particular to the Background Source(s)
▪ Implement Techniques to Reduce or Eliminate Unwanted Staining
2019 Annual Tri-State Plus One Histology Symposium 2 Introduction
Overview PART 1: Immunohistochemistry
▪ Background / Issues ▪ IHC Workflow ▪ Sources of Background ▪ Strategies to Eliminate Background ▪ Summary
PART 2: Immunofluorescence
2019 Annual Tri-State Plus One Histology Symposium 3 What is Background?
Unwanted non-specific staining observed on the test specimen. ▪ Trace ▪ Moderate ▪ Severe
Arise from one or more sources: ▪ Inherent to the specimen ▪ Detection reagents ▪ Combination of tissue & reagents
Oxidized/polymerized substrate reaction product deposited on the specimen.
2019 Annual Tri-State Plus One Histology Symposium 4 Problems Arising From Background Staining
Assay Validation ▪ What is specific?
Interference with specific staining ▪ Confusion with antigen expression
False positives ▪ Staining in same cell type
Goal: Clear, Unambiguous View of Target Antigen
2019 Annual Tri-State Plus One Histology Symposium 5 Background – Now What?
2019 Annual Tri-State Plus One Histology Symposium 6 IHC Workflow
Step 1: Tissue Preparation/Antigen Retrieval
Step 2: Quenching / Blocking
Step 3: Primary Antibody Incubation
Step 4: Secondary Detection Reagent
Step 5: Substrate / Chromogen
Step 6: Counterstain / Mount
Step 7: Visualize
2019 Annual Tri-State Plus One Histology Symposium 7 IHC Workflow
Step 1: Tissue Preparation/Antigen Retrieval – Protocol Optimization
Step 2: Quenching / Blocking
Step 3: Primary Antibody Incubation
Step 4: Secondary Detection Reagent Main Focus
Step 5: Substrate / Chromogen
Step 6: Counterstain / Mount
2019 Annual Tri-State Plus One Histology Symposium 8 Identify the Source(s) of the Background
Run Defined Controls ▪ Positive (Protocol Optimization) ▪ Negative (Deletions)
Key Steps in One-Step Polymer IHC Detection Procedure / Workflow
Primary Antibody Incubation Enzyme Polymer Incubation React with Substrate
2019 Annual Tri-State Plus One Histology Symposium 9 But …..
“..nothing has changed.”
2019 Annual Tri-State Plus One Histology Symposium 10 Well something has …
1. Antigen Retrieval Protocol Optimization 2. Times, Temperature, Washes (Positive Control)
3. Heterogenous Tissue Block ▪ Cell Type ▪ Antigen Expression ▪ Vascularization ▪ Inflammation ▪ Necrosis
2019 Annual Tri-State Plus One Histology Symposium 11 First Deletion Negative Control
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)
2019 Annual Tri-State Plus One Histology Symposium 12 Staining with First Deletion Control
Issues arising from: ▪ Lack of quenching (omitted; diluted/old reagent; time) ▪ Inappropriate enzyme (HRP or AP) quench
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).
2019 Annual Tri-State Plus One Histology Symposium 13 Quenching Endogenous Enzyme Activity in Tissue Sections
WITHOUT WITH
Presence of endogenous peroxidase activity indicated by application of substrate (DAB) alone, no other detection reagents. Acetone-fixed frozen tonsil sections, hematoxylin counterstain (blue).
2019 Annual Tri-State Plus One Histology Symposium 14 Quenching Endogenous Enzyme Activity in Tissue Sections
WITHOUT WITH
Presence of endogenous alkaline phosphatase activity indicated by application of substrate (Fast Red) alone, no other detection reagents. Acetone-fixed frozen tonsil sections, hematoxylin counterstain (blue).
2019 Annual Tri-State Plus One Histology Symposium 15 Quenching Methods
Endogenous peroxidase:
1) Hydrogen peroxide (H2O2) – Most commonly used ▪ 3% H2O2 in water Home brew / ▪ 0.3% in Methanol (MeOH) Commercial
2) Alternative methods: ▪ Andrew S.M., Jasani, B. (1987) Histochem J. 19: 426-430. ▪ Malorny, U. et al (1988) J. Immunol. Meth. 111(1):101-107
2019 Annual Tri-State Plus One Histology Symposium 16 Quenching Methods
Endogenous Alkaline Phosphatase: Primarily an issue for frozen sections as the AP enzyme is heat labile, hence not as greater an issue for paraffin embedded material.
1) Add Levamisole (1 – 5 mM) to AP Substrate Working Solution. 2) Acidic solution ▪ Ponder, B. A. and Wilkinson, M. M. (1981) J. Histochem. Cytochem. 29(8):981-984
2019 Annual Tri-State Plus One Histology Symposium 17 Enzyme Substrates
1) Not All (HRP/AP) Substrates Are Equal
Differences: ▪ Concentration ▪ Reaction kinetics Varying Sensitivity Could Generate ▪ Solvents Excessive Color Deposition (background) ▪ Stability of working solution
2) Converting from HRP to AP (vice versa) Requires Re-Optimization
3) Be Aware of Inherent Pigment and Tissue Elements Interpreted Melanin in Tissue Section as “Background” (Hematoxylin Counterstain)
2019 Annual Tri-State Plus One Histology Symposium 18 Second Deletion Negative Control
Apply Substrate & Enzyme Conjugated Detection Reagent (No primary antibody)
▪ Treat section(s) as per SOP. ▪ Just prior to applying primary antibody, apply HRP polymer. ▪ Apply (DAB) substrate for usual time. ▪ Do not counterstain (i.e. no hematoxylin). ▪ View. Background staining in Absence of Primary Antibody. (Note counterstain).
Determines Whether Detection Reagent is Binding Non-Specifically (Not Sourced from Specimen – Introduced/Exogenous Source)
2019 Annual Tri-State Plus One Histology Symposium 19 Staining with Second Deletion Control
Issues Attributed to:
▪ Species cross-reactivity ▪ Inadequate blocking ▪ Inadequate buffer washes ▪ Concentration too high ▪ Decrease incubation time
Background staining in absence of primary antibody (Hematoxylin counterstain)
2019 Annual Tri-State Plus One Histology Symposium 20 Staining with Second Deletion Control
Species Cross-Reactivity: Without Specific Mouse Ig Block ▪ Species on species ➢ Mouse on mouse / Xenograft model ✓ Commercial (block) options ✓ Change primary antibody species ✓ Conjugate primary antibody
With Specific Mouse Ig Block ▪ Closely related species ➢ Anti-mouse IgG on rat tissue (vice versa) ✓ Use pre-adsorbed reagent ✓ Dilute in serum from specimen
2019 Annual Tri-State Plus One Histology Symposium 21 Staining with Second Deletion Control
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.
2019 Annual Tri-State Plus One Histology Symposium 22 Staining with Second Deletion Control
Other Contributing Factors:
▪ Inadequate buffer washes ▪ Concentration too high Protocol Optimization Check with Positive Control Data ▪ Decrease incubation time
Shorter Wash Time Longer Wash Time Adjacent sections exposed to varying buffer wash times after polymer incubation step. Hematoxylin Counterstain.
2019 Annual Tri-State Plus One Histology Symposium 23 Staining with Second Deletion Control
Avidin/Biotin (non-polymer) based Deletion Controls ▪ Substrate Only ▪ Substrate & (Strept)Avidin enzyme Conjugate ➢ 0.05% avidin + 0.005% biotin in buffer for effective blocking ▪ Substrate & (Strept)Avidin enzyme Conjugate & Biotinylated Secondary Key Steps in Two-Step (Strept)Avidin/Biotin IHC Detection Procedure
Primary Antibody Biotinylated Enzyme conjugated React with Substrate Incubation Secondary Incubation (Strept)Avidin / ABC
2019 Annual Tri-State Plus One Histology Symposium 24 Complete Detection System
Inappropriate Staining ▪ Ensure primary antibody has been validated (commercial source / purified?) ➢ Recommended guidelines: ✓ Titer ✓ Time Check positive ✓ Temperature control tissue ✓ 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.
2019 Annual Tri-State Plus One Histology Symposium 25 What Is An Appropriate Negative Control?
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.
Simply omitting the primary antibody
2019 Annual Tri-State Plus One Histology Symposium 26 Further Tools and Resources
Resource Tools IHC Literature Texts / Journals Workflows Product Selection Guides Troubleshooting Guides Vendor Websites Protocols Data Sheets Technical Support Video Tutorials JOVE YouTube
2019 Annual Tri-State Plus One Histology Symposium 27 Summary
GOAL: Clear, Unambiguous View of Target Antigen
Implement Adjustments
Troubleshooting
Negative Controls
Positive Controls
Familiar with IHC Workflow
2019 Annual Tri-State Plus One Histology Symposium 28 Questions?
2019 Annual Tri-State Plus One Histology Symposium 29 Introduction
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
2019 Annual Tri-State Plus One Histology Symposium 30 Fluorescence
Definition: Absorption of electromagnetic radiation at one wavelength and re-emission at another lower energy wavelength.
Fluorescein (or FITC) Absorption 492 nm Emission 515 nm Wavelength (nm)
2019 Annual Tri-State Plus One Histology Symposium 31 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)
2019 Annual Tri-State Plus One Histology Symposium 32 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.
Figure: http://zeisscampus.magnet.fsu.edu/ articles/lightsources/metalhalide.html
2019 Annual Tri-State Plus One Histology Symposium 33 Sources of Background Fluorescence
Unwanted non-specific fluorescence observed on the test specimen.
Arise from one or more sources: ▪ Inherent to the specimen – “Autofluorescence” ▪ Detection reagents – e.g. Primary / Secondary Antibodies ▪ Combination of tissue & reagents Fluorescent compounds endogenous and/or introduced on the specimen
Don’t be like this guy. Get a clue and identify the background source(s)
2019 Annual Tri-State Plus One Histology Symposium 34 Basic IF Workflow
Untreated Step 1: Tissue Preparation (Antigen Retrieval)
Step 2: Protein Blocking
Step 3: Primary Antibody Incubation
Step 4: Secondary Antibody Fluorophore Conjugated Sodium borohydride Sudan Black B Step 5: Coverslip with Antifade Medium
Step 6: Visualize
2019 Annual Tri-State Plus One Histology Symposium 35 IF Workflow
Step 1: Tissue Preparation (Antigen Retrieval)
Step 2: Protein Blocking ▪ Protocol Optimization Step 3: Primary Antibody Incubation ▪ Positive Controls ▪ Negative Controls Step 4: Secondary Detection Reagent As per IHC Step 5: Coverslip / Visualize
Background Fluorescence Arising from the Detection Reagents
2019 Annual Tri-State Plus One Histology Symposium 36 What is Autofluorescence?
Autofluorescence:
Unwanted, background fluorescent signal arising from endogenous tissue components and/or induced through Collagen the use of a fixative. Adversely affects signal to noise ratio.
Three main sources: Formalin fixation Red blood cells 1. Lipofuscin – tissue pigment/granules
2. Aldehyde fixed material (i.e. formalin, paraformaldehyde) 3. Endogenous tissue elements (RBCs, Collagen)
2019 Annual Tri-State Plus One Histology Symposium 37 Tissue Autofluorescence
Fixation:
▪ Formalin/formaldehyde ➢ Most common for IF, leads to blue and green fluorescence (FFPE)
▪ Glutaraldehyde ➢ Leads to more extensively crosslinked tissue and higher fluorescence in yellow and red spectral region
2019 Annual Tri-State Plus One Histology Symposium 38 Endogenous Fluorescence
Endogenous fluorescence that is particularly problematic in animal tissue
Source Fluorescent Channels Reason Red Blood Cells Green and Red Hemoglobin and (RBCs) Oxidative Stress Connective Tissue Blue, Green and Red Protein Cross-Links (elastin, collagen) Lipofuscin Green and Red Lipid Cross-Links
2019 Annual Tri-State Plus One Histology Symposium 39 Endogenous Fluorescence
Figure: Monici, M. (2005) Biotechnol. Ann. Rev., 11:227-256
Fluorescent molecules, protein side chains, protein cross-links: NAD(P)H, chlorophyll, porphyrins, collagen, elastin, retinol, tyrosine, phenylalanine, tryptophan, flavin, pyridoxine, indoleamine, melanin, lipofuscin
2019 Annual Tri-State Plus One Histology Symposium 40 Endogenous Fluorescence
A retinal lipofuscin fluorophore molecule
Conjugated double bonds
Aromatic group
2019 Annual Tri-State Plus One Histology Symposium 41 Differences Among Tissue Types
Fixed tissue More problematic than frozen
Spleen, Kidney, Pancreas Especially problematic for autofluorescence
Brain Tissue Significant amount of fluorescence due to lipofuscin; punctate bright signals
2019 Annual Tri-State Plus One Histology Symposium 42 Far-Red and Near-IR Dyes
Far-Red Dyes: Used to avoid the autofluorescence of tissue elements
Figure: http://www.biomedima.org/?modality=5&slide=396
2019 Annual Tri-State Plus One Histology Symposium 43 Conventional Solutions
▪ Chemical treatments
▪ Photo treatments
▪ Masking treatments
▪ Quenching treatments
2019 Annual Tri-State Plus One Histology Symposium 44 Chemical Treatments
▪ Potassium permanganate (KMnO4); oxidative reaction
▪ Hydrogen peroxide (H2O2); oxidative reaction
▪ Sodium borohydride (NaBH4); reductive reaction
▪ Glycine; addition reaction
▪ Ammonia/ethanol; addition reaction
2019 Annual Tri-State Plus One Histology Symposium 45 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
2019 Annual Tri-State Plus One Histology Symposium 46 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
2019 Annual Tri-State Plus One Histology Symposium 47 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
▪ Trypan Blue: somewhat effective in flow cytometry; introduces far-red fluorescence
▪ Eriochrome Black T: somewhat effective for FFPE tissue
2019 Annual Tri-State Plus One Histology Symposium 48 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.
2019 Annual Tri-State Plus One Histology Symposium 49 Previous Investigations
Untreated Eriochrome Black T
FFPE Human Tracheal Tissue.
Reference: Sodium Borohydride Sudan Black B Davis, et al., (2014) J. Histochem Cytochem, 62(6):405-423
2019 Annual Tri-State Plus One Histology Symposium 50 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.
2019 Annual Tri-State Plus One Histology Symposium 51 Vendor AF Quenching Products
Company Product Target Compound
MaxVision MaxBlock AFR Kit Lipofuscin & Ink based Aldehyde induced Neuromics FluoMute AF Blocking Lipofuscin & CuS04 based Agent Aldehyde induced Millipore/Sigma AutoFlu. Eliminator Lipofuscin Ink based Reagent Biotium TrueBlack Lipofuscin Ink based
“Home Brews” Target Compound Sodium Borohydride Aldehyde Induced Chemical based
Sudan Black Lipofuscin Ink based
2019 Annual Tri-State Plus One Histology Symposium 52 Reduction of Lipofuscin
Ink based reagents are effective against lipofuscin.
Differences in effectiveness may be apparent across the spectrum.
Imaged sourced directly from Biotium website: https://biotium.com/product/trueblack- lipofuscin-autofluorescence-quencher/
2019 Annual Tri-State Plus One Histology Symposium 53 Latest Advances
Most RecentUntreated 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
▪ ShortSodium Treatment borohydride (2-5 minutes)Sudan Black B ▪ Short Wash After Treatment (2-5 minutes) ▪ Can Be Scaled Up For Increased Slide Volume
2019 Annual Tri-State Plus One Histology Symposium 54 Comparison of Autofluorescence Reduction (AFR) Treatments
UntreatedUntreated Human Pancreas
Collagen TrueVIEW
Formalin fixation Red blood cells Sodium borohydride Copper Sulfate Sudan Black B Sodium Borohydride
2019 Annual Tri-State Plus One Histology Symposium 55 Mode of TrueVIEW Treatment
Untreated Specific signal (fluorophore labelled secondary antibody)
Fluorescent endogenous “elements”
AFR Treatment Sodium borohydride Sudan Black B
Specific signal retained, with background signal reduced
2019 Annual Tri-State Plus One Histology Symposium 56 Human Spleen
Without Treatment With AFR Treatment
Autofluorescence in TrueVIEW Treatment green channel
Sodium borohydride
Adjacent human spleen tissue sections double stained using primary antibodies against CD20 (red) and Ki67 (green) antigens. Left, untreated; right treated. White arrowsAutofluorescence indicated true antigen in staining. TrueVIEW Treatment red channel 2019 Annual Tri-State Plus One Histology Symposium 57 If You Encounter AF …
2019 Annual Tri-State Plus One Histology Symposium 58 …Glove Up and Pinpoint the Source
AF Source Treatment
Tissue elements and/or TrueVIEW AFR use of aldehyde fixative (Vector)
MaxBlock AFR Kit (MaxVision)
FluoMute AF Blocking Agent (Neuromics) Lipofuscin AutoFlu. Eliminator Reagent (Millipore/Sigma)
TrueBlack (Biotium)
2019 Annual Tri-State Plus One Histology Symposium 59 Conclusions
1) Beyond the detection reagents, fixation and endogenous tissue elements can introduce significant autofluorescence (AF) background in an immunofluorescence (IF) assay.
2) Determine the source of the AF – lipofuscin, tissue elements, aldehyde induced.
Sodium3) Once borohydride the source has been identified, apply appropriate measures to reduce the AF.
4) Run assay again and ensure AF has been reduced in the part(s) of the spectrum you are targeting. Autofluorescence in red channel 2019 Annual Tri-State Plus One Histology Symposium 60 Questions?
Autofluorescence in TrueVIEW Treatment green channel
Sodium borohydride
Autofluorescence in TrueVIEW Treatment red channel 2019 Annual Tri-State Plus One Histology Symposium 61 Thank you
Craig Pow, Ph.D. Director, Technical Service [email protected]
2019 Annual Tri-State Plus One Histology Symposium