6-plex IF Protocol on the BOND RX (Leica Biosystems)

Multiplex IF assay on the Bond RX Autostainer (Leica Biosystems) using the Opal multiplex IHC system (PerkinElmer/Akoya Biosciences Cat# NEL871001KT) and the BOND Polymer Refine Detection Kit (Leica Biosystems Cat# DS9800).

• Prepare the antibody and the fluorophore dilutions in 6mL Leica BOND tubes (see Ficial M et al for details about reagents, dilutions and order of antibodies).

• Fill an empty 7ml Leica BOND container with DAPI.

• Fill an empty 30ml Leica BOND container with Dako Protein Block.

Bake the unstained FFPE slides in the Isotemp Oven for 30 mins at 60°C.

Deparaffinization and Rehydration

• BOND Dewax Solution (Leica Biosystems Cat# AR9222)

Time. (min): 30:00, Temperature: 72°C, Dispense type: open.

• Rehydration.

Bond ER Solution 1 (Leica Biosystems Cat# AR9961; citrate, pH 6.0)

Time. (min): 10:00, Temperature: 98°C, Dispense type: 150 μL.

Deionized Water

Time. (min): 2:00; Temperature: Ambient; Dispense type: 150 μL.

Bond Wash Solution (Leica Biosystems Cat# AR9590)

Time. (min): 2:00; Temperature: Ambient; Dispense type: 150 μL.

Peroxide Block (Leica Biosystems Cat# DS9800)

Time. (min): 10:00; Temperature: Ambient; Dispense type: 150 μL.

Bond Wash Solution (Leica Biosystems Cat# AR9590)

Time. (min): 4:00; Temperature: Ambient; Dispense type: 150 μL.

Dako Protein Block (Agilent Cat# X090930-2)

Time. (min): 15:00; Temperature: Ambient; Dispense type: 150 μL.

Primary Antibody (diluted in either Leica Biosystems BOND Primary Antibody Diluent Cat# AR9352 or Biocare Medical DaVinci Green Diluent Cat# PD900 M)

Time. (min): 40:00; Temperature: Ambient; Dispense type: 150 μL.

Bond Wash Solution (Leica Biosystems Cat# AR9590)

Time. (min): 4:00; Temperature: Ambient; Dispense type: 150 μL.

Post-Primary (Leica Biosystems Cat# DS9800) *

Time. (min): 15:00; Temperature: Ambient; Dispense type: 150 μL.

*Post-Primary should be used after mouse anti-human primary antibodies only; when using rabbit anti-human primary antibodies, please go directly to step 14.

For TIM-3 this step was replaced by:

Rabbit anti Goat Link

Time. (min): 15:00; Temperature: Ambient; Dispense type: 150 μL

Bond Wash Solution (Leica Biosystems Cat# AR9590)

Time. (min): 4:00; Temperature: Ambient; Dispense type: 150 μL.

Polymer (Leica Biosystems Cat# DS9800)

Time. (min): 10:00; Temperature: Ambient; Dispense type: 150 μL.

Bond Wash Solution (Leica Biosystems Cat# AR9590)

Time. (min): 6:00; Temperature: Ambient; Dispense type: 150 μL.

Opal Fluorophore (PerkinElmer/Akoya Biosciences Cat# NEL871001KT)

Time. (min): 5:00; Temperature: Ambient; Dispense type: 150 μL.

Bond Wash Solution (Leica Biosystems Cat# AR9590)

Time. (min): 6:00; Temperature: Ambient; Dispense type: 150 μL.

Bond ER Solution 1(Leica Biosystems Cat# AR9961; citrate, pH 6.0)

Time. (min): 10:00, Temperature: 98°C, Dispense type: 150 μL.

Start a new cycle of staining from step 5 OR go to step 20

Last cycle of staining:

Deionized Water

Time. (min): 2:00; Temperature: Ambient; Dispense type: 150 μL.

DAPI (PerkinElmer/Akoya Biosciences Cat# NEL871001KT)

Time. (min): 10:00; Temperature: Ambient; Dispense type: 150 μL.

Manually coverslip using Invitrogen ProLong Diamond Antifade Mountant (Thermo Fisher Scientific Cat# P36962).