1x Plus Amplification Diluents

Overview

These ready-to-use diluents have been optimized specifically for Opal™ and TSA® Plus immunohistochemistry protocols.

Catalog

Details

Utilize these diluents to bring your Opal fluorophores to working concentration.
1X Plus Manual Amplification Diluents are for use with Opal fluorophores when staining manually, typically at 1:100 dilution. The 1X Plus Automation Amplification Diluent is formulated specifically for Opal multiplex IHC fluorophores used on the Leica BOND RX, for use at 1:150.

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 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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