strategies to identify and interference in ihc and if tissue · key steps in two-step...
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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
Craig Pow, Ph.D. Director, Technical Service
16th Annual Tri-State Plus One Histology Symposium
Des Moines, IA May 8-10, 2019
2019 Annual Tri-State Plus One Histology Symposium
Objectives
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Upon completion of this workshop, participants will be responsible to:
▪ Identify Sources of Non-Specific Staining in Tissue Based Assays
▪ Evaluate Options Particular to theBackground Source(s)
▪ Implement Techniques to Reduce orEliminate Unwanted Staining
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Overview
PART 1: Immunohistochemistry
▪ Background / Issues▪ IHC Workflow▪ Sources of Background▪ Strategies to Eliminate
Background▪ Summary
PART 2: Immunofluorescence
Introduction
2019 Annual Tri-State Plus One Histology Symposium
What is Background?
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Unwanted non-specific stainingobserved 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.
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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
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Background – Now What?
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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
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IHC Workflow
Step 2: Quenching / Blocking
Step 3: Primary Antibody Incubation
Step 4: Secondary Detection Reagent
Step 5: Substrate / Chromogen
Step 6: Counterstain / Mount
Main Focus
Step 1: Tissue Preparation/Antigen Retrieval – Protocol Optimization
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.
Acetone-fixed frozen tonsil sections, hematoxylin counterstain (blue).
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WITHOUT WITH
Quenching Endogenous Enzyme Activity in Tissue Sections
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).
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Quenching Methods
Endogenous peroxidase:
1) Hydrogen peroxide (H2O2) – Most commonly used
▪ 3% H2O2 in water
▪ 0.3% in Methanol (MeOH)
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
Home brew / Commercial
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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
Enzyme Substrates
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1) Not All (HRP/AP) Substrates Are Equal
Differences:▪ Concentration▪ Reaction kinetics▪ Solvents ▪ Stability of working solution
Varying Sensitivity Could Generate Excessive Color Deposition (background)
2) Converting from HRP to AP (vice versa) Requires Re-Optimization
3) Be Aware of Inherent Pigment and Tissue Elements Interpreted as “Background”
Melanin in Tissue Section(Hematoxylin Counterstain)
2019 Annual Tri-State Plus One Histology Symposium
Second Deletion Negative Control
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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.
Determines Whether Detection Reagent is Binding Non-Specifically(Not Sourced from Specimen – Introduced/Exogenous Source)
Background staining in Absence of Primary Antibody. (Note counterstain).
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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)
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Species Cross-Reactivity:
▪ Species on species ➢ Mouse on mouse / Xenograft model
✓ Commercial (block) options✓ Change primary antibody species✓ Conjugate primary antibody
▪ 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
▪ 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
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:
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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.
Fluorescein (or FITC)Absorption 492 nmEmission 515 nm Wavelength (nm)
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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.
Figure: http://zeisscampus.magnet.fsu.edu/articles/lightsources/metalhalide.html
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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Step 2: Protein Blocking
Step 3: Primary Antibody Incubation
Step 4: Secondary Detection Reagent
Step 5: Coverslip / Visualize
Step 1: Tissue Preparation (Antigen Retrieval)
▪ Protocol Optimization ▪ Positive Controls ▪ Negative Controls
As per IHC
IF Workflow
Background Fluorescence Arising from the Detection Reagents
2019 Annual Tri-State Plus One Histology Symposium
Autofluorescence:
Unwanted, background fluorescent signal arising from endogenous tissue components and/or induced through the use of a fixative.
Adversely affects signal to noise ratio.
Three main sources:
1. Lipofuscin – tissue pigment/granules
2. Aldehyde fixed material (i.e. formalin, paraformaldehyde)
3. Endogenous tissue elements (RBCs, Collagen)
What is Autofluorescence?
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Red blood cells
Formalin fixation
Collagen
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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
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Endogenous Fluorescence
Source Fluorescent Channels Reason
Red Blood Cells (RBCs)
Green and Red Hemoglobin and Oxidative Stress
Connective Tissue (elastin, collagen)
Blue, Green and Red Protein Cross-Links
Lipofuscin Green and Red Lipid Cross-Links
Endogenous fluorescence that is particularly problematic in animal tissue
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Endogenous Fluorescence
Fluorescent molecules, protein side chains, protein cross-links:
NAD(P)H, chlorophyll, porphyrins, collagen, elastin, retinol, tyrosine, phenylalanine, tryptophan, flavin, pyridoxine, indoleamine, melanin, lipofuscin
Figure: Monici, M. (2005) Biotechnol. Ann. Rev., 11:227-256
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Endogenous Fluorescence
A retinal lipofuscin fluorophore molecule
Conjugateddouble bonds
Aromaticgroup
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Differences Among Tissue Types
Fixed tissue More problematic than frozen
Spleen, Kidney, Pancreas Especially problematic for autofluorescence
Brain TissueSignificant amount of fluorescence due to lipofuscin; punctate bright signals
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Far-Red and Near-IR Dyes
Figure: http://www.biomedima.org/?modality=5&slide=396
Far-Red Dyes: Used to avoid the autofluorescence of tissue elements
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Conventional Solutions
▪ Chemical treatments
▪ Photo treatments
▪ Masking treatments
▪ Quenching treatments
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Chemical Treatments
▪ Potassium permanganate (KMnO4); oxidative reaction
▪ Hydrogen peroxide (H2O2); oxidative reaction
▪ Sodium borohydride (NaBH4); reductive reaction
▪ Glycine; addition reaction
▪ Ammonia/ethanol; addition reaction
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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
▪ Trypan Blue: somewhat effective in flow cytometry; introduces far-red fluorescence
▪ 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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Vendor AF Quenching Products
Company Product Target Compound
MaxVision MaxBlock AFR Kit Lipofuscin & Aldehyde induced
Ink based
Neuromics FluoMute AF Blocking Agent
Lipofuscin & Aldehyde induced
CuS04 based
Millipore/Sigma AutoFlu. Eliminator Reagent
Lipofuscin Ink based
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
Reduction of Lipofuscin
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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/
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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
Thank you
Craig Pow, Ph.D. Director, Technical Service