Manual

Multiplex IHC
Research Use Only. Not for use in diagnostic procedures.

Opal™ 4 Lymphocyte Kit

Product Information
Storage

Store dry Opal reagent at -20 ºC. Upon reconstituting in DMSO, store at 2–8ºC.
Store remaining kit components at 2–8ºC

Stability

See kit label on outside of box for expiration date.

Application

The Opal 4 Lymphocyte Kit is intended for detection of CD 4, CD8 and CD20 on tumor FFPE
sections. The Opal 4 Lymphocyte Kit is optimized for manual use.

Species Reactivity

Human.

Safety Note

Some reagents in this kit contain DMSO that is classified as hazardous and combustible. Some®
reagents in this kit contain Proclin 300 that is classified as corrosive to metals and skin, a skinand eye irritant, and hazardous to the aquatic environment. DAPI is considered corrosive to theskin and an irritant to the eye. All other reagents are classified as nonhazardous. It is strongly
recommended to wear disposable gloves and safety glasses while working with the items in thiskit. Thorough washing of hands after handling is also recommended.

Quality Control

We certify that QC results of these reagents meet our quality release criteria.

What is the Opal Method?
The Opal workflow allows simultaneous detection of multiple biomarkers in tissue. This Opal protocol was written
specifically for 4-Color IHC in formalin fixed paraffin embedded (FFPE)
tissue to detect three lymphocyte markers on tumor FFPE sections.*
The approach involves detection with Opal reactive fluorophores,
followed by microwave treatment (MWT) for: removal of primary and1secondary antibodies ; removal of any non-specific staining; andreduction of tissue auto-fluorescence. The Opal signal is largely
unaffected by MWT and antibody removal. After MWT, another round of
staining can be performed for additional target detection without risk ofantibody cross reactivity.

Human lung cancer tissue imaged on the
Mantra™ quantitative pathology workstation.
Target
CD4
CD8
CD20
DAPI

Color
Green
Cyan
Red
Blue

Opal allows staining of multiple IHC targets using unlabeled primary2
antibodies raised in the same species . Combining Opal withmultispectral imaging and analysis enables simultaneous, quantitative
results for up to 6 biomarkers in fluorescence, even with co-localized
markers, plus nuclear counterstain (DAPI).
*Please contact us if you would like to work with other types ofsamples. PerkinElmer provides assistance with assay development
and offers multiplex Opal IHC and IF services. Visit:www.perkinelmer.com/Opal.
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Description

Format*Opal 4 Lymphocyte Kit

50 slides

Catalog #Kit Components
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OP4LY2001KT

1X Plus Amplification Diluent (1X 25mL)
Opal 520 Fluorophore
Opal 570 Fluorophore
Opal 670 Fluorophore
Spectral DAPI solution
DMSO (1 X 500 µL)10X AR6 buffer (1 X 250ml)
10X AR9 buffer (1 X 250ml)
Antibody Diluent (1 X 50ml)
Anti-CD4, anti-CD8, anti-CD20, antibodies(human reactivity)
Opal Polymer HRP Ms + Rb (25ml)*The format of the kit is based on ~150 µL per slide of Opal Working Solution (see page 4).

Reagents and Materials
Required Materials
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Required ReagentsBaths and solvents for deparaffinization andrehydration of FFPE tissue. Xylene isrecommended for deparaffinization. Histologicalgrade ethanol is required for rehydration.
Standard microwave oven with carousel, rated at
1000W or higher with 10 or more power settings.
Standard staining dishes.
Opal Slide Processing Jars (STJAR4).
Slide incubation/humidity tray.
Hydrophobic barrier pen.
Orbital platform shaker (optional).
Glass coverslips.
Control tissues.
Charged Slides.

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10% Neutral buffered formalin (NBF)
TBST wash buffer.
Peroxidase-free water.
Note: This specification may be met bycommercial “cell culture grade” water or ultra-pure(i.e. Milli-Q™) water that has been autoclaved.
Mounting medium for fluorescenceSolutions to prepare
TBST Wash Buffer
25 mM TRIS-HCl, pH 7.5
150 mM NaCl
0.05% Tween®20 (v/v)
AR6 Buffer Working Solution:
Dilute 10X AR6 buffer at 1:10 with peroxidase-free water.
AR9 Buffer Working Solution:
Dilute 10X AR9 buffer at 1:10 with peroxidase-free water.
Antibody Diluent
Antibody Diluent / Block from PerkinElmer comes as a ready-to-use solution.
Primary Antibody Working Solution
Dilute primary antibody in Antibody Diluent / Block at optimal concentration for Opal detection as determined below.
Secondary Antibody Working Solution
Opal Polymer HRP Ms + Rb comes as a ready-to-use solution and does not need to be optimized for use with Opal
fluorophores.
Opal Fluorophore Working Solution
Dissolve the Opal Reagents in 75µL of DMSO. Carefully dispense DMSO along the sides of the vial several times to dissolve
any Opal Reagent that might coat the sides of the vial. Minimize bubbles while mixing.
Before each procedure, dilute Opal Fluorophore 1:50 in 1X Amplification Diluent to make Opal Fluorophore Working Solution.
Generally, 150 µL of Opal Working Solution is required per slide. Discard any unused portion of Opal Working Solution.

DAPI working solution
Add two drops of DAPI solution into 1ml of TBST. Approximately 150 µL of DAPI Working Solution is required per slide.
Discard any unused portion of DAPI Working Solution.

Recommendations
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Use xylene for removal of paraffin from FFPE tissue sections. Do not let slides dry out between steps.
Before each use, spin down the Opal Fluorophore tubes and primary antibody tubes with a standard microcentrifuge
to make sure that all of the solution is at the bottom of the tubes.
A humidified chamber is recommended for all incubation steps (i.e., a damp paper towel in a covered box).
Drain off as much of the incubation solutions as possible before addition of the next solution, to prevent reagent
dilution and uneven staining. Blot area around, but not on, tissue section using absorbent paper.
Be sure to use enough volume of each reagent to completely cover sections or cells.
Optimized exposure times on the Mantra or Vectra systems for all fluorophores should be between 50 msec and 200
msec for good spectral unmixing.
Spectral DAPI in this kit is formulated for optimal separation from other fluorophores. Exposure time may besomewhat longer than other DAPI formulations.
If there is too much signal, dilute the primary antibody further.
Before attempting multiplexed staining, assay conditions for each analyte should be optimized singly with Opal
detection.
Microwave treatment (MWT) as outlined in this protocol performs antigen retrieval, quenches endogenous
peroxidases, and removes antibodies from earlier staining procedures.
This protocol was developed with specified reagents. Other options should be independently validated.
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Opal Optimization Strategies
Microwave Optimization
Microwave treatment (MWT) is used in the Opal method to quench endogenous peroxidase activity, for antigen retrieval,and to remove antibodies after a target has been detected. Timing for each step in the procedure may have to bemodified for the microwave oven that you are using. Slides are placed vertically in an Opal Slide Processing Jar which isthen filled to the top (~140 mL) with AR6 or AR9 buffer and covered loosely. One jar is placed in the microwave at a time,near the edge of the carousel to ensure even distribution of energy. The microwave procedure consists of two steps.
1.
100% power until the boiling point is reached. The time for this step may have to be increased or decreased
depending upon the performance of the microwave in your lab. This will usually take 45-90 seconds.
2.
20% power for 15 minutes.
Do not operate the microwave oven unattended and keep the oven chamber clean and clear of debris.

Primary Antibody Optimization
Opal Monoplex IHC assays should be optimized individually before combining for use in multiplexed detection. The
optimal primary antibody concentration will result in highest signal to background ratio observed in fluorescencemicroscopy, with roughly similar signal levels for each target. Vectra and Mantra autoexposure times for optimized assays
should fall between 50 and 200 msec for all targets.
Follow the suggested optimization steps below:
 Slide 1: use the suggested dilution rate in table 1.
 Slide 2-3: use half log (3.16-fold) serial dilutions from suggested dilution. Further dilution or less dilution thansuggested dilution may be necessary.
 Slide 4: negative control (primary antibody omitted).

Table 1: Suggested starting dilution for primary antibodies
Antigen retrieval

Antibody

Suggested final
concentration (ng/ml)

Incubation time

Opal Fluorophore

AR9
AR9
AR6

CD4
CD8
CD20

70
120
100

30 min30 min30 minOpal 520
Opal 570
Opal 670

If autoexposure times are too long under evaluated conditions, consider extending the incubation time or increasing the
primary antibody concentration.
Reduce the antibody concentration if autoexposure times are less than 50 msec. Excessive signal can prevent unmixingof fluorescent spectra.

Slide Definitions
Successful optimization of a multiplex assay requires three different types of slides.
• Monoplex. Control tissue slides are labeled for one marker with a single Opal fluorophore and counterstained with
DAPI. They are necessary for assessment of staining results and comparison to established standards. This may
come from the primary antibody optimization.
• Library. Spectral library slides stained with a single fluorophore and made from relevant control tissue will benecessary for accurate unmixing and analysis of your monoplex and multiplex slides. The following slides will be
required.
o One control tissue slide stained for each antibody-Opal fluorophore combination under optimized
conditions, without DAPI. Three single stained slides: CD4-Opal520, CD8-Opal570 and CD20-Opal670
stained slides without DAPIo One control tissue slide stained with DAPI alone.
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One unstained control tissue slide, for assessment of autofluorescence. The unstained slides should beprocessed in the same way as the other slides, omitting both the Opal fluorophore and DAPI.
Multiplex. Tissue slides are labeled for all of the markers in the multiplex panel and counterstained with DAPI. These
slides allow assessment of the multiplex assay for interference and crosstalk.

Step by Step Opal IHC Protocol
Please consult the Opal Assay Development Guide prior to beginning an Opal Multiplex
Protocol: http://www.perkinelmer.com/category/cancer-immunotherapy-immunology
The full Opal 4-color protocol generally takes 1 day to complete. Here are some points where the procedure may bepaused.
After microwave treatment, cooling in AR buffer at room temperature.
0Store in Antibody Diluent or blocking buffer at 4 C.
0
Overnight primary antibody incubation at 4 C. (Antibody incubation conditions must be validated for the assay.)0
Store slides in TBS or TBST overnight at 4 C after each staining circle.

Opal Monoplex Protocol
1. Deparaffinization. Heat slides in a dry oven at 55-60 °C for four hours, positioned to allow drainage of melting paraffin.
Wash slides with xylene for 10 minutes, 3 times. Hydrate through an ethanol gradient ending with a distilled water wash.
2. Slide Fixation. Fix tissue in NBF for 20 minutes followed by a distilled water wash.
3. Antigen Retrieval/Microwave treatment (MWT). Rinse slides with AR6 or AR9 solution as appropriate for the target
being detected. Place slides in an Opal slide processing jar and fill with AR6 or AR9 solution immediately to prevent theslides from drying out. Perform MWT under optimized conditions as determined previously and allow slides to cool down
to room temperature on the bench for at least 15 minutes.
4. Blocking. Rinse slides with TBST. Encircle the area of tissue to be stained with a hydrophobic barrier pen. Incubate
the tissue with Antibody Diluent for 10 minutes at room temperature.
5. Primary Antibody Incubation. Remove the blocking solution and then apply the primary antibody solution to thetissue. Antibody concentration and incubation time must be optimized for Opal detection.
6. Secondary Antibody Incubation. Apply the secondary antibody solution to the tissue. Opal Polymer HRP should be
incubated for 10 minutes at room temperature. Wash slides with TBST 3 times, 2 minutes each.
7. Opal Fluorophore Incubation. Apply Opal fluorophore solution to the tissue and incubate for 10 minutes at room
temperature. Wash slides with TBST 3 times, 2 minutes each.
8. Antibody Removal. Rinse slides twice with AR6 solution. Place slides in an Opal slide processing jar with AR6
solution and fill up to the top. Perform MWT and allow slides to cool down to room temperature on the bench for at least
15 minutes.
9. Spectral DAPI. Rinse slides with TBST. Incubate slides in DAPI solution for 5 minutes at room temperature. Wash
slides with TBST for 2 minutes, and then with distilled water for 2 minutes. (Skip this step where appropriate for single
stain library slides.)10. Mount. Apply mounting medium for fluorescence microscopy and coverslip.

Opal Multiplex Protocol
Optimized Opal Monoplex IHC methods may be combined for multiplexed detection within a single tissue section. Aftersignal amplification, MWT is performed to strip away detection antibodies. The Opal fluorophore is largely unaffected by
MWT because it is covalently bound. Then the process is repeated using another Opal fluorophore as outlined below.
A checklist to follow as you perform the protocol is on page 9.

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1. Deparaffinization. Heat slides in a dry oven at 55-60 °C for four hours, positioned to allow drainage of melting paraffin.
Wash slides with xylene for 10 minutes, 3 times. Hydrate through an ethanol gradient ending with a distilled water wash.
2. Slide Fixation. Fix tissue in NBF for 20 minutes followed by a distilled water wash.
3. Multiplex staining of six markers:

Cycle 1:C1.1. Antigen Retrieval/MWT. Rinse slides with AR9 solution. Place slides in an Opal slide processing jar with
AR9 solution and fill up to the top. Perform MWT under optimized conditions as determined previously andallow slides to cool down to room temperature on the bench for at least 15 min.
C1.2. Blocking. Rinse slides with TBST. Encircle the area of tissue to be stained with a hydrophobic barrier
pen. Incubate the tissue with antibody diluent for 10 min at room temperature (RT).
C1.3. Primary Antibody Incubation. Remove the blocking solution and then apply the anti-CD4 antibody
solution to the tissue using the optimized concentration and incubation time determined previously .Wash slides
with 1X TBST for 3 times at 2 min each.
C1.4. Secondary Antibody Incubation. Apply Opal Polymer HRP anti-Ms/Rb secondary antibody solution to
the tissue and incubate for 10 min at RT. Wash slides with 1X TBST for 3 times, 2 min each.
C1.5. Opal Fluorophore Incubation. Apply Opal-520 working solution to the tissue and incubate for 10 min at
RT . Wash slides with TBST 3 times, 2 minutes each.
C1.6. Microwave Treatment. Rinse slides with AR9. Place slides in an Opal slide processing jar with AR9solution and fill up to the top. Perform MWT as previously and allow slides to cool down to room temperature on
the bench for at least 15 minutes.

Cycle 2:C2.1. Blocking. Rinse slides with TBST. Encircle the area of tissue to be stained with a hydrophobic barrier
pen. Incubate the tissue with Antibody Diluent for 10 min at room temperature (RT).
C2.2. Primary Antibody Incubation. Remove the blocking solution and then apply the anti-CD8 antibody
solution to the tissue using the optimized concentration and incubation time determined previously .Wash slides
with 1X TBST for 3 times at 2 min each.
C2.3. Secondary Antibody Incubation. Apply Opal Polymer HRP anti-Ms/Rb secondary antibody solution to
the tissue and incubate for 10 min at RT. Wash slides with 1X TBST for 3 times, 2 min each.
C2.4. Opal Fluorophore Incubation. Apply Opal-570 working solution to the tissue and incubate for 10 min at
RT . Wash slides with TBST 3 times, 2 minutes each.
C2.5. Microwave Treatment. Rinse slides with AR6. Place slides in an Opal slide processing jar with AR6
solution and fill up to the top. Perform MWT as previously and allow slides to cool down to room temperature on
the bench for at least 15 minutes.

Cycle 3:C3.1. Blocking. Rinse slides with TBST. Encircle the area of tissue to be stained with a hydrophobic barrier
pen. Incubate the tissue with Antibody Diluent for 10 min at room temperature (RT).
C3.2. Primary Antibody Incubation. Remove the blocking solution and then apply the anti-CD20 antibody
solution to the tissue using the optimized concentration and incubation time determined previously .Wash slides
with 1X TBST for 3 times at 2 min each.
C3.3. Secondary Antibody Incubation. Apply Opal Polymer HRP anti-Ms/Rb secondary antibody solution to
the tissue and incubate for 10 min at RT. Wash slides with 1X TBST for 3 times, 2 min each.
C3.4. Opal Fluorophore Incubation. Apply Opal-670 working solution to the tissue and incubate for 10 min at
RT . Wash slides with TBST 3 times, 2 minutes each.
C3.5. Microwave Treatment. Rinse slides with AR6. Place slides in an Opal slide processing jar with AR6
solution and fill up to the top. Perform MWT as previously and allow slides to cool down to room temperature on
the bench for at least 15 minutes.
4. Spectral DAPI. Rinse slides in distilled water and then in TBST. Incubate slides in DAPI solution for five minutes atroom temperature. Wash slides with TBST for two minutes, and then with distilled water for two minutes.

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5. Mount. Apply mounting medium for fluorescence microscopy and coverslip.

Imaging and Analysis
Visualization of 4-color Opal slides can be performed using standard fluorescent microscopy using the suggested filter
sets shown below.
Fluorophore

Excitation

Emission

Suggested Filter Set

Spectral DAPI

358 nm461 nmDAPI

Opal 520

494 nm

517 nmFITC

Opal 570

550 nm

570 nmCY3

Opal 670

648 nm

667 nmCY5

Multispectral imaging allows quantitative unmixing of many fluorophores and removal of tissue autofluorescence, enablingadvanced analysis including in situ cellular phenotyping. For more information, please see:
http://www.perkinelmer.com/quantitative-pathology.
Important Notes for imaging with Vectra or Mantra:
1. All standard Vectra or Mantra epi-fluorescent cubes should be used for imaging Opal slides: DAPI, FITC, CY3,
Texas Red, CY5.
2. If the Opal fluorophores are not found in the Stain Store Manager in your version of inForm, please visit
http://www.perkinelmer.com/resources/software-downloads.xhtml to download the latest inForm update that
contains the novel Opal stains. If you believe that you have the latest version of inForm and you still cannot findthe stains in the store manager, please contact your Field Application Specialist.

Please see the Phenoptics Assay Development Guide for more detail on imaging and analysis.

References
1Toth, Zsuzsanna E., and Eva Mezey. "Simultaneous visualization of multiple antigens with tyramide signal amplification
using antibodies from the same species." Journal of Histochemistry & Cytochemistry 55.6 (2007): 545-554
2

Stack, E.C., Wang, C., Roman, K., and Hoyt, C.C. “Multiplexed immunohistochemistry, imaging, and quantitation: areview, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.” Methods:(2014) doi:10.1016/j.ymeth.2014.08.016.

Troubleshooting
Technical Support Resources Email: Telephoneo USA toll-freeo Worldwide
o Fax
o Local contact numbers:

global.techsupport@perkinelmer.com
800-762-4000+1 203-925-4602+1 203-944-4904
http://www.perkinelmer.com/corporate/locations

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Related Products
Opal Fluorophore Excitation and Emission Maxima
Wavelength

Fluorophore

Cap color

Excitation

EmissionSpectral DAPI

358nm

461 nmBlue

Opal 520

494 nm

525nm

Green

Opal 540

523nm

536nm

Yellow

Opal 570

550 nm

570 nmRed

Opal 620

588 nm

616 nmAmber

Opal 650

627 nm

650 nmOrangeOpal 690

676nm

694 nmClear

Related Products
Opal Multiplex IHC Detection Kits
SIZES

PRODUCT NUMBER

Opal 4-Color Automation IHC Kit

50 slides

NEL820001KT

Opal 7-Color Automation IHC Kit

50 slides

NEL821001KT

Opal 4-Color Manual IHC Kit

50 slides

NEL810001KT

Opal 7-Color Manual IHC Kit

50 slides

NEL811001KT

Opal 4 Lymphocyte Kit

50 slides

OP4LY2001KT

Opal 7 Immunology Discovery Kit

50 slides

OP7DS2001KT

Opal 7 Tumor Infiltrating Lymphocyte Kit

50 slides

OP7TL3001KT

Opal 7 Solid Tumor Immunology Kit

50 slides

OP7TL4001KT

Opal Reagent Packs
PRODUCT NUMBER
Opal 520 Reagent Pack

FP1487001KT

Opal 540 Reagent Pack

FP1494001KT

Opal 570 Reagent Pack

FP1488001KT

Opal 620 Reagent Pack

FP1495001KT

Opal 650 Reagent Pack

FP1496001KT

Opal 690 Reagent Pack

FP1497001KT

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Ancillary
PRODUCT NUMBER
1X Plus Amplification Diluent 1 X 25 mL

FP1498A

AR6 buffer (10X) 4 x 250 mL

AR6001KT

AR6 buffer (10X) 250 mL

AR600250ML

AR9 buffer (10X) 4 x 250 mL

AR9001KT

AR9 buffer (10X) 250 mL

AR900250ML

Antibody Diluent / Block 100 mL

ARD1001EA

Opal Polymer HRP Ms + Rb 50 mL

ARH1001EA

Spectra DAPIFP1490A

For the latest product listings, please go to www.perkinelmer.com/opal.

TSA and its use are protected under U.S. Patents 5,688,966, 5,863,748, 5,767,287, 6,372,937, 6,399,299, 6,593,100, 6,617,125 and patents pending, and foreign equivalents thereof.
Research use only. This product is distributed and sold for research purposes only by the end-user in the research market, and, to that extent, by purchasing this product the end-user is granted a limited
license to use this product for research use only. This product is not intended for diagnostic or therapeutic use and no license or right is granted for use of this product for diagnostic or therapeutic
purposes. Purchase does not include or carry any right or license to use, develop or otherwise exploit this product commercially. Any commercial use, development or exploitation of this product without
the express prior written authorization of PerkinElmer is strictly prohibited and may constitute infringement of the intellectual property rights of PerkinElmer under the aforementioned patents.
TSA is a registered trademark of PerkinElmer. Opal is a trademark of PerkinElmer. Other trademarks are property of their respective owners.

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Quick Start Guide / Protocol Checklist

Opal 4 Lymphocyte Panel A Kit
o Multiplex slide√Marker 1
CD4√

Marker 2
CD8√

Marker 3
CD20√

Counter Stain
DAPI

Deparaffinization & Fixation
AR/MWT

AR9

AR9

AR6

AR6

Wash

TBST

TBST

TBST

TBST

Blocking

10min @RT

10min @RT

10min @RT

Anti-CD4

Anti-CD8

Anti-CD20

Antibody/CounterStain

DAPI

Dilution
Inc.Time

5min @ RT

Inc.Temp.
Wash

TBST

TBST

TBST

Opal HRP Polymer Ms+ Rb

10min @ RT

10min @ RT

10min @ RT

Wash

TBST

TBST

TBST

Opal fluor

Opal 520

Opal 570

Opal 670

Incubation

10min @ RT

10min @ RT

10min @ RT

Wash

TBST

TBST

TBST

Spectral library slideso Six single stained slides: CD4-Opal 520, CD8-Opal 570, CD20-Opal 670 stained slides without DAPIo One control tissue slide stained with DAPI alone.
o One AF slide processed in the same way as the multiplex slide, omitting both the Opal fluorophore and DAPI
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