AKOYA FP1494001KT 540 Reagent Pack Instructions

FP1494001KT 540 Reagent Pack
Instructions

FP1494001KT 540 Reagent Pack

AUTOMATED/MANUAL | Multiplex IHC
OpalTM 540 Reagent Pack FP1494001KT
MATERIAL PROVIDED

• Opal 540, 1 vial
• DMSO, 1 x 100 uL

Please note that Opal 540 is provided dry. DMSO has been included to reconstitute this dye. Please follow the protocol provided below.

PRODUCT INFORMATION

Protocol| • Dissolve the Opal 540 Reagent in 75 uL 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.
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Storage/Stability| Long Term Storage – Store Lyophilized reagents in the dark at -20 ºC.
Short Term Storage – Performance is not impacted when lyophilized reagents are stored at room
temperature (RT) for less than seven days.
• Reconstituted reagent in DMSO or ddH20 must be store in the dark at 4oC and must be used
within three months when stored as directed.
• Working solutions must be stored at 4°C, minimizing light exposure. DO NOT FREEZE.
• Working solutions are good for 24-48 hours when stored at 4°C.
• See label on outside of the box for expiration date.
Safety Note| DMSO is classified as hazardous and combustible. 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.
Opal Fluorophore Working Solution| Before each procedure, dilute Opal fluorophore in 1X Plus Amplification Diluent (product number FP1498) to make Opal Fluorophore working solution. if manually staining. If staining on an automated platform, dilute Opal fluorophore in 1X Plus Automation Amplification Diluent (product number FP1609). The following dilution ratios are recommended:
• 1:100 for manual
• 1:150 for automated
Generally, 100-300 μL of Opal working solution is required per slide. Discard any unused portion of Opal working solution.

PROCEDURE

Refer to Opal Assay Development Guide for more detailed instruction:
Single analyte Opal IHC assays should be optimized before combining for use in multiplexed detection. Concentration for each primary antibody should be optimized with  the selected Opal fluorophore to yield an exposure time of 50 – 250ms or have an intensity between 5 to 30. Optimized single fluorophore images (without DAPI counterstain will subsequently be used for spectral library development. 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 each experiment. Bake each slide in the oven at 65oC 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 (NBF) for 20min. Longer times of fixation may be needed for certain tissues such as skin.
Rinse slides in distilled water and then in the appropriate buffer.
STEP 2: MICROWAVE TREATMENT
Place slides in an Opal Staining Jar (product number STJAR4) 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 Tris Buffered Saline, 0.1% Tween 20 (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 Akoya Biosciences Antibody Diluent/Block (product number ARD1001EA) 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 enough volume 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 Opal Polymer HRP Ms + Rb (product number ARH1001EA) secondary antibody for 10 min at room temperature. Use adequate reagent volume to cover the tissue section, generally 100-300 μL per slide.
• Note: Opal Polymer HRP Ms + Rb is recommended for experiments with human tissue and mouse or rabbit primary antibodies. Other options should be independently 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 working solution onto each slide. Incubate the slides 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.
Rinse slides in the appropriate AR buffer.
STEP 7: MICROWAVE TREATMENT
Place slides in an Opal Staining 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; optimization may be required 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 (Thermo Fisher Scientific)). (Note: do not counterstain monoplex slides to be used for spectral library
development.)
Imaging and Analysis
Visualization of slides stained with Opal 540 can be performed using PhenoImager™ HT (formerly Vectra® Polaris™ ) and Mantra 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: https://www.akoyabio.com/phenoimager

Troubleshooting

https://www.akoyabio.com/support/ 
support@akoyabio.com

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.
For 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 enduser 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 Akoya Biosciences is strictly prohibited and may constitute infringement of the intellectual property rights of Akoya Biosciences under the aforementioned patents.
TSA is a registered trademark of Akoya Biosciences. Opal is a trademark of Akoya Biosciences. Other trademarks are property of their respective owners

To learn more visit AKOYABIO.COM or email us at INFO@AKOYABIO.COM
For Research Use Only. Not for use in diagnostic procedures.
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All trademarks are the property of Akoya Biosciences unless otherwise specified.
DN-00085 Rev A

References

• AKOYABIO.CO
• PhenoImager Solution | Akoya Biosciences
• Technical and Application Support | Akoya Biosciences

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