AKOYA BIOSCIENCES FP1495001KT Opal 620 Reagent Pack Instruction Manual
- June 4, 2024
- AKOYA BIOSCIENCES
Table of Contents
AKOYA BIOSCIENCES FP1495001KT Opal 620 Reagent Pack Instruction Manual
PRODUCT INFORMATION
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 asdescribed}. 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 620 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/
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.
© 2022 Akoya Biosciences, Inc. All rights reserved. All trademarks are the
property of Akoya Biosciences unless otherwise specified.
DN-00082 Rev A
References
- The Spatial Biology Company | Akoya Biosciences
- PhenoImager Solution | Akoya Biosciences
- Technical and Application Support | Akoya Biosciences