Manual

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

Opal™ 4-Color anti-Rabbit Manual IHC Kit 50 slides

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

This product is stable for a minimum of 3 months when stored at 2–8 °C

Application

The Opal 4-Color anti-Rabbit Manual IHC Kit is intended for multiplex fluorescent IHC.

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

Quality Control

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

Slide Number

When using this kit’s recommended Opal dilution of 1:100, it will enable a 4-color assay on 50
slides.

What is the Opal Method?
The Opal workflow allows simultaneous detection of multiple biomarkers
in tissue. This Opal protocol was written specifically for a 4 or 7-Color
IHC in formalin-fixed paraffin-embedded (FFPE) tissue. The approach
involves detection with Opal fluorophores, followed by microwave
treatment (MWT) for: removal of primary and secondary antibodies1;
removal of any non-specific staining; and reduction of tissue autofluorescence. 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 of antibody cross reactivity.
Opal allows staining of multiple IHC targets using unlabeled primary
antibodies raised in the same species2. 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). Fluorescent multispectral imaging (usually with the Mantra™ or Vectra®
systems) is required for successful analysis of more than 3 Opal fluorophores at once.

PerkinElmer provides assistance with assay development and offers multiplex Opal IHC and IF services. Visit:www.perkinelmer.com/Opal.

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Material Provided
Format*Opal 4-Color anti-Rabbit
Manual IHC Kit

50 slides

Catalog #Kit Components
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NEL840001KT

1X Plus Amplification Diluent (1 X 50 mL)
Opal 520 Fluorophore
Opal 570 Fluorophore
Opal 690 Fluorophore
DMSO ( 1 X 500 µL)
Spectral DAPI solution (1 X 1.5 mL)
Opal Polymer anti-Rabbit HRP ( 1 X 10mL)
Opal Polymer anti-Rabbit HRP Diluent (1x 40mL)
Blocking/Ab Diluent (1 X 100 mL)
10X AR6 buffer (2 X 250ml)*The format of the kit is based on ~150 µL per slide of Opal Working Solution.

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
1,000 W or higher with 10 or more power settings
Standard staining dishes
Opal slide processing jars (Perkin Elmer catalogue
number STJAR4) or equivalent
Slide incubation/humidity tray
Hydrophobic barrier pen
Glass coverslips (No. 1.5)
Control tissues
Charged slides

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TBST wash buffer
Neutral buffered formalin (NBF)
Peroxidase-free water.
Note: This specification may be met bycommercial “cell culture grade” water or ultra-pure(i.e. Milli-Q™) water.
Antibody diluent & blocking reagento Antibody Diluent / Block (PerkinElmer
catalog number ARD1001EA) is
recommendedo Mouse tissue often requires more robust
blocking methods, such as goat or horse
serum.
o Other options should be validated
independently
Primary antibodies for targets of interest
Mounting medium for fluorescence (i.e. ProLong®
Diamond Antifade Mountant (Thermofisher)).
AR9 Buffer (PerkinElmer catalog number
AR900250ML) may be required for certain antigens
requiring a higher pH antigen retrieval buffer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.
Antibody Diluent
Antibody Diluent / Block from PerkinElmer is supplied as a ready-to-use solution.
Primary Antibody Working Solution
Dilute primary antibody in PerkinElmer Antibody Diluent / Block at optimal concentration for Opal detection as determined
below.
Secondary Antibody Working Solution
Opal Polymer anti-Rabbit HRP is supplied as a concentrate. Working dilution recommendation for manual assays is 1:5 inthe supplied Opal Polymer anti-Rabbit HRP Diluent.
Opal Reagent Stock Solution
Dissolve the Opal Reagents in 75 uL of DMSO. Carefully dispense DMSO along the sides of the vial several times todissolve any Opal Reagent that might coat the sides of the vial. ( Minimize bubbles while mixing.)
Opal Fluorophore Working Solution
Before each procedure, dilute Opal Fluorophore in 1X Amplification Diluent to make Opal Fluorophore Working Solution.
Recommend to start diluting the Opal fluorophore at 1:100. Optimize your assay according on Opal Assay Development
Guide. Generally, 100-300 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• Use xylene for removal of paraffin from FFPE tissue sections. Do not let slides dry out between steps.
• Before the first use, spin down the Opal Fluorophore 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.
• 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.
• Optimize your monoplex staining according to the Opal Assay Development Guide, with all MWT steps incorporated.
• Spectral DAPI in this kit is formulated for optimal separation from other fluorophores. Exposure time may besomewhat longer than other DAPI formulations.
• Before attempting multiplexed staining, assay conditions for each analyte should be optimized singly with Opal
detection (refer to Opal Assay Development Guide for more details).
• 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.
• If the antigen requires higher pH retrieval, it is recommended to purchase PerkinElmer’s AR9 Buffer.
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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 unattended and keep the oven chamber clean and clear of debris.

Opal Multiplexed IHC
Single analyte Opal IHC methods may be combined for multiplexed detection within a single tissue section. After signal
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.
Importantly, a different Opal fluorophore should be used for each target.

Opal Workflow Schematic

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Step by Step Opal IHC Protocol (Single Analyte) Refer to Opal Assay Development Guide formore detailed instruction:
Single analyte Opal IHC assays should be optimized before combining for use in multiplexed detection. Concentration foreach primary antibody should be optimized with the selected Opal fluorophore to yield intensity between 10 to 30 counts.
Optimized single fluorophore images (without DAPI counterstain) will subsequently be used for spectral librarydevelopment.
The following protocol details the workflow for a single analyte, and can subsequently be employed for multiplexed IHC.
In multiplexed IHC, the order of target/fluorophore detection may be a point of optimization, and must be independently
validated.
Step 1: Slide Preparation
Prepare tissues or cells for detection with Opal using standard fixation and embedding techniques. We recommend
running an isotype control slide with all steps replacing the primary antibody with corresponding isotype control for eachexperiment. For each slide, baked in the oven at 65⁰C for 1 hour; dewax with xylene (3 x 10 min) and rehydrate through a
graded series of ethanol solutions: (100% 1 x 10 min; 95% 1 x 10 min; and rinse in 70%). After rehydration, briefly rinse
slides in distilled water and fix in 10% neutral buffered formalin for 20min. Longer times of fixation in NBF may be needed
for certain tissues such as skin.
Rinse slides in distilled water and then in the appropriate AR buffer.

Step 2: Microwave treatment
Place slides in an Opal Slide Processing Jar and fill it completely with the appropriate AR buffer. Loosely cover the jar
with lid, place it in microwave for 45 sec at 100% power; (may require optimization as described). Microwave for an
additional 15 min at 20% power. Allow slides to cool down at room temperature before proceeding (15 – 30 min).
Importantly, do not let slides dry out. Rinse slides in distilled water followed by TBST.

Step 3: Blocking
Use a hydrophobic barrier pen to completely surround the tissue section on the slide. Cover tissue sections with blocking
buffer and incubate slides in a humidified chamber for 10 min at room temperature.
• Note: This protocol was developed with PerkinElmer Antibody Diluent / Block for blocking. Other options should
be independently validated.

Step 4: Primary Antibody Incubation
Drain off the blocking buffer and apply Primary Antibody Working Solution. Incubate according to the manufacturer's
instructions regarding incubation time and temperature requirements or conditions optimized within your lab. Use enoughvolume to completely cover the tissue section (generally 100-300 µL per slide).
Rinse slides in TBST. Wash the slides 3 x 2 min in TBST at room temperature, preferably with agitation.
Step 5: Introduction of Secondary-HRP
Incubate slides in the working solution Opal Polymer anti-Rabbit HRP for 10 min at room temperature. Use adequate
reagent volume to cover the tissue section, generally 100-300 µL per slide.
• Note: Opal Polymer anti-Rabbit HRP working concentration is recommended at 1:5 with the provided Opal
Polymer Diluent. Other concentrations need to be validated.
Rinse slides in TBST. Wash the slides 3 x 2 min in TBST at room temperature preferably with agitation.
Step 6: Opal Signal Generation
Drain off excess wash buffer and pipette 100-300 µL of Opal Fluorophore Working Solution onto each slide. Incubate theslides at room temperature for 10 mins.
Rinse slides in TBST. Wash the slides 3 x 2 min in TBST at room temperature preferably with agitation.
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Rinse slides in the appropriate AR buffer.
Step 7: Microwave treatment
Place slides in an Opal Slide Processing Jar and fill it completely with the appropriate AR buffer. Loosely cover the jar
with lid, place it in microwave for 45 sec at 100% power; (may require optimization as described). Microwave for an
additional 15 mins at 20% power. Allow slides to cool down at room temperature before proceeding (15 – 30 min).
Importantly, do not let slides dry out. Rinse slides in distilled water followed by TBST.
This microwave step strips the primary-secondary-HRP complex allowing introduction of the next primary antibody. For
detection of the next target, restart the protocol at Step 3: Blocking.
If all targets have been detected, continue to Step 8.
Step 8: Counterstain and Mount
Apply DAPI Working Solution for 5 min at room temperature in a humidity chamber. Wash the slides for 2 min in TBST
buffer and then for 2 min in water. Coverslip slides with mounting medium (i.e. ProLong® Diamond Antifade Mountant(Thermofisher)). (Note: do not counterstain monoplex slides to be used for spectral library development.)

Imaging and Analysis
Visualization of 4- or 7-color Opal slides can be performed using Mantra or Vectra Quantitative Pathology Imaging
Systems. The systems use multispectral imaging for quantitative unmixing of many fluorophores and tissue
autofluorescence, enabling advanced analysis including in situ cellular phenotyping. For more information, please see:
http://www.perkinelmer.com/quantitative-pathology.
Important Notes:
1. All standard Vectra or Mantra epi-fluorescent cubes should be used for imaging Opal slides: DAPI, FITC, CY3,
Texas Red, and 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.

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.

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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

IHC Troubleshooting
PROBLEM

REMEDY

Low Signal••

Excess Signal

High
Background•

Titer primary and Opal dye to determine optimum concentration for Opal detection.
Increase primary antibody and Opal Working Solution incubation time. (Suggested range
is 3-10 minutes.)
Use additional rounds of microwave treatment to unmask the target.

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Decrease concentration of primary antibody.
Decrease concentration of Opal dyes
Decrease Opal Working Solution incubation time. (Suggested range is 3-10 minutes.)
Confirm that microwave treatment step has fully quenched endogenous peroxidases.
Titer primary and/or Opal dyes to determine optimum concentration for Opal detection.
Freshly prepare buffers.
Evaluate laboratory water source for contamination with HRP.
Check for endogenous biotin (if using streptavidin conjugates)
Increase number and/or length of washes.

Opal Fluorophore Excitation and Emission Maxima
Opal Multicolor IHC Kits

Fluorophore

Excitation

Emission

Cap color

✓

358nm

461 nm

Blue

✓

✓

494 nm

525nm

Green

Opal540



✓

523nm

536nm

Yellow

Opal570✓✓

550 nm

570 nm

Red

Opal620✓

588 nm

616 nmAmber✓

627 nm

650 nmOrange✓

676nm

694 nmClear

4-color7-colorSpectral DAPI✓

Opal520

Opal650
Opal690✓

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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*

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NEL821001KT

Opal 4-Color Manual IHC Kit

50 slides

NEL810001KT

Opal 7-Color Manual IHC Kit

50 slides

NEL811001KT

Opal 4-Color anti-Rabbit Automation IHC Kit* 50 Slides

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NEL840001KT

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OP7TL4001KT*Optimized for Leica Biosystems BOND RX System

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

Ancillary
PRODUCT NUMBER
1X Plus Automation Amplification Diluent 1 X 50 mL

FP16091X Plus Amplification Diluent 1 x 50 mL

FP1498

Opal Polymer anti-Rabbit HRP Kit

ARR1001KT

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

ARD1001A

Opal Polymer HRP Ms + Rb 50 mL

ARH1001A

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

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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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