Opal Fluorophore Reagent Packs

Overview

Opal™ is a practical workflow for simultaneous detection of up to 8 tissue biomarkers plus nuclear counterstain, within a single image. The method is similar to standard immunohistochemistry and is accessible to many laboratories where standard IHC method development is performed. Standalone Opal Fluorophore reagent packs allow you to pick and choose your panel’s biomarker quantity and design.

Catalog

Details

Each Opal Reagent Pack contains 1 vial DMSO and enough Opal reagent for up to 50 slides in a multiplex IHC protocol.

Opal 780 is a two-step antibody-mediated reaction and requires special considerations, please refer to the product technical data sheet.

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

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