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

Opal™ 4-Color anti-Rabbit Automation IHC Kit 50 slides
Optimized for Leica Biosystems BOND RX System
Product Information
Storage

Store kits in the dark at 4ºC.

Stability

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

Application

The Opal 4-Color anti-Rabbit Automation IHC kit is intended for multiplex fluorescent IHC.

User Manual

Download the latest version of the User Manual at www.perkinelmer.com/opal

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. Allother reagents 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.

®

What is the Opal Method?
The Opal workflow allows simultaneous detection of multiple
biomarkers in tissue. This Opal protocol was written specificallyfor 4 or 7-Color IHC on formalin fixed paraffin embedded (FFPE)
tissue section*. The approach involves detection with Opal
reactive fluorophores, followed by heat-induced
stripping for: removal of primary and secondary antibodies; removalof any non-specific staining; and reduction of tissue autofluorescence. The Opal signal is largely unaffected by stripping andantibody removal. After stripping step, another round of staining canbe 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 species. Combining Opal withmultispectral imaging and analysis enables simultaneous,quantitative results for up to 6 biomarkers influorescence, 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 morethan 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
Automation IHC Kit

50 slides

Catalog #NEL830001KT

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

Usage Instructions
Dilute Opal Polymer anti-Rabbit HRP to the working concentration with provided Opal Polymer anti-Rabbit Diluent.
Recommended dilution: 1:5
Other dilutions should be validated.

Troubleshooting
Technical Support Resources• Email:global.techsupport@perkinelmer.com• Telephoneo USA toll-free
800-762-4000
o EU toll-free
00800 33 29 0000
o China toll-free
800 820 5046
o Local contact numbers: http://www.perkinelmer.com/corporate/locations

IHC Troubleshooting
PROBLEM
REMEDY
Low Signal•••Excess Signal••••••••High Background

Titer primary and/or Opal dyes to determine optimum concentration for Opal detection.
Increase primary antibody and/or Opal Working Solution incubation time.
Change staining order of multiplex and move this specific target detection towards the end of
staining procedure..
Decrease concentration of primary antibody.
Decrease concentration of Opal dyes.
Decrease Opal Working Solution incubation time. (Suggested range is 3-10 minutes.)
Add extra step to block endogenous peroxidases.
Titer primary and/or Opal dyes to determine optimum concentration for Opal detection.
Make fresh buffers.
Evaluate laboratory water source for contamination.
Increase number and/or length of washes.

Opal Fluorophore Excitation and Emission Maxima
Opal Multicolor IHC Kits

Fluorophore

4-colorExcitation

Emission

Cap color

7-colorSpectral DAPI

✓

✓

358nm

461 nmBlue

Opal520✓✓

494 nm

525nm

GreenOpal540



✓

523nm

536nm

Yellow

Opal570✓✓

550 nm

570 nm

RedOpal620

✓

588 nm

616 nmAmber✓

627 nm

650 nmOrangeOpal6502
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Opal690✓✓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-Color anti-Rabbit Automation IHC Kit* 50 Slides

NEL830001KT

Opal 4-Color anti-Rabbit Manual IHC Kit

50 Slides

NEL840001KT

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

SIZES

PRODUCT NUMBER

Opal520 Reagent, powder

50 slides

FP1487A

Opal540 Reagent, powder

50 slides

FP1494A

Opal570 Reagent, powder

50 slides

FP1488A

Opal620 Reagent, powder

50 slides

FP1495A

Opal650 Reagent, powder

50 slides

FP1496A

Opal690 Reagent, powder

50 slides

FP1497A

Spectra DAPI

50 slidesFP1490A*Optimized for Leica Biosystems BOND RX System

Opal Reagents

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.

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 therapeutic3
1-05-40-NR-OPAL-NEL830 Rev-01

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 intellec tual 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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