Spectral DAPI is a nuclear counterstain for use with Opal Multiplex IHC assays.
The Opal Reagent Packs can be utilized for both mono- and multiplex tissue staining and have been configured and validated for imaging on the Vectra® and Mantra™ systems.
Opal Multiplex IHC Kits make multiplex methods accessible to anyone who works with standard immunohistochemistry. The Opal method allows use of any number of unlabeled primary antibodies from the same species in multiplexed tissue assays, with no fear of cross-reactivity. Antibodies for simultaneous IHC may be selected based on performance, rather than species. The method works with formalin fixed paraffin-embedded (FFPE) tissue and is compatible with the standard research IHC workflow in your lab. Note: If you have never used Opal Multiplex IHC Detection Reagents before, we strongly suggest purchasing the full Opal kit, which includes a set of quality controlled reagents that have been optimized to work together.
Specifications
Catalog Number
Shipping Condition
Blue Ice
Sample Species
Human and Mouse
Product Category
Opal
Unit Size
50 Slides
Wavelength
368/461 nm
Type
Reagent Pack
Detection Method
Vectra Polaris
Automation Compatible
Yes
Workflow
Bake and Deparaffinize Slides
- Bake at 60 C for one hour
- Xylene wash (3x ten minutes)
- Rehydrate with EtOH gradient into di H2O
Slide Fixation
- 10% NBF for 20 minutes
- Di H2O wash
Antigen Retrieval
- AR6 or AR9 microwave treatment (MWT)
- Cool to RT for at least 15 minutes
Blocking
- Di H2O rinse, TBST rinse
- PAP pen barrier
- Incubate tissue for 10 minutes in blocking solution at RT
Primary Antibody
- Remove blocker and add primary antibody
- Rinse with TBST, then wash 3 times for two minutes each in TBST
Secondary Antibody
- Incubation 10 minutes at RT
- Rinse with TBST and wash 3 times for 2 minutes each
Opal Reagent
- Opal fluorophore incubation for ten minutes at RT
- Rinse with TBST and wash 3 times for 2 minutes each
MWT
- Rinse slides with AR6 or AR9
- Place slides in microwave-safe jar, perform MWT
- Cool to RT for at least 15 minutes
Spectral DAPI
- Use AR6 for MWT prior to DAPI for removal of any unbound fluor
- Rinse slides in di H2O and then TBST
- Incubate DAPI for 5 minutes at RT
- Wash with di H2O and TBST two minutes each
Mount
- Apply monunting medium for fluorescene microscopy for coverslip
Documents
Education Resources
OPAL Multiplex for Tissue Phenotyping – A Lab Guide
Multispectral Imaging to Detect Immune Phenotypes within the Tumor Microenvironment
A fully optimized end-to-end solution for I/O multiplex immunofluorescence staining using Opal Polaris 7-Color PD1/PD-L1 Panel Kits for Lung Cancer and Melanoma
Applying multispectral unmixing and spatial analyses to explore tumor heterogeneity with a pre-optimized 7-color immuno-oncology workflow