10X buffer for use in Opal™ immunohistochemistry multiplexing protocols. This buffer was developed for use in Opal multicolor immunohistochemistry protocol in microwave treatment (MWT) steps. MWT provides antigen retrieval for formalin-fixed, paraffin embedded (FFPE) tissue, quenches endogenous peroxidase, and removes antibodies after epitopes have been labeled with Opal dyes.
Specifications
Catalog Number
AR900250ML
Shipping Condition
Blue Ice
Product Category
Opal
Unit Size
250 mL
Type
Buffer
Detection Method
Vectra Polaris
Automation Compatible
No
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
Multispectral Imaging to Detect Immune Phenotypes within the Tumor Microenvironment
Multispectral Imaging to Detect Immune Phenotypes within the Tumor Microenvironment
OPAL Multiplex for Tissue Phenotyping – A Lab Guide
OPAL Multiplex for Tissue Phenotyping – A Lab Guide
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
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
Applying multispectral unmixing and spatial analyses to explore tumor heterogeneity with a pre-optimized 7-color immuno-oncology workflow