Strategies To Identify and Interference in IHC and IF Tissue · Key Steps in Two-Step...

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]

16th Annual Tri-State Plus One Histology Symposium

Des Moines, IA May 8-10, 2019


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

2019 Annual Tri-State Plus One Histology Symposium 3

Overview

PART 1: Immunohistochemistry

▪ Background / Issues▪ IHC Workflow▪ Sources of Background▪ Strategies to Eliminate

Background▪ Summary

PART 2: Immunofluorescence

Introduction


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.


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


Background – Now What?


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


IHC Workflow

Step 2: Quenching / Blocking



Step 5: Substrate / Chromogen

Step 6: Counterstain / Mount

Main Focus

Step 1: Tissue Preparation/Antigen Retrieval – Protocol Optimization


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


But …..

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“..nothing has changed.”


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


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)


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).


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).


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).


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


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


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)


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).


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)


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



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.



▪ 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


Other Contributing Factors:


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


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


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


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


Further Tools and Resources

Resource Tools

IHC Literature Texts / JournalsWorkflowsProduct Selection Guides Troubleshooting Guides

Vendor Websites ProtocolsData SheetsTechnical Support

Video Tutorials JOVEYouTube


Familiar with IHC Workflow

Positive Controls

Negative Controls

Troubleshooting

Implement Adjustments

Clear, Unambiguous View of Target Antigen

Summary

GOAL:


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


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)


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)


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


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)


Basic IF Workflow

Untreated

Sodium borohydride Sudan Black B

Step 1: Tissue Preparation (Antigen Retrieval)

Step 2: Protein Blocking


Step 4: Secondary Antibody Fluorophore Conjugated

Step 5: Coverslip with Antifade Medium

Step 6: Visualize


Step 2: Protein Blocking



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


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


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


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



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



A retinal lipofuscin fluorophore molecule

Conjugateddouble bonds

Aromaticgroup


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


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


Conventional Solutions

▪ Chemical treatments

▪ Photo treatments

▪ Masking treatments

▪ Quenching treatments


Chemical Treatments

▪ Potassium permanganate (KMnO4); oxidative reaction

▪ Hydrogen peroxide (H2O2); oxidative reaction

▪ Sodium borohydride (NaBH4); reductive reaction

▪ Glycine; addition reaction

▪ Ammonia/ethanol; addition reaction


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


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


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


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.


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


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.


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


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/

https://biotium.com/product/trueblack-lipofuscin-autofluorescence-quencher/

https://biotium.com/product/trueblack-lipofuscin-autofluorescence-quencher/


Latest Advances

Untreated


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


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


Mode of TrueVIEW Treatment

Untreated


Specific signal (fluorophore labelled secondary antibody)

Fluorescent endogenous “elements”

AFR Treatment

Specific signal retained, with background signal reduced


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


If You Encounter AF …

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…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


Conclusions

Sodium borohydride


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.


Sodium borohydride

Autofluorescence in green channel


TrueVIEW Treatment

TrueVIEW Treatment

Questions?


Thank you

Craig Pow, Ph.D. Director, Technical Service

[email protected]

Strategies To Identify and Interference in IHC and IF Tissue · Key Steps in Two-Step...

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