Jul 12, 2022

Public workspace6-plex IF Protocol on the BOND RX (Leica Biosystems)

DOI
dx.doi.org/10.17504/protocols.io.bjbzkip6
  • 1Department of Pathology, Brigham and Women’s Hospital, Boston, MA, 02115, USA;
  • 2Harvard Medical School, Boston, MA, 02215, USA
Miriam Ficial
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DOI: dx.doi.org/10.17504/protocols.io.bjbzkip6
Protocol Citation: Maura A Sticco-Ivins, Abdallah Flaifel, Miriam Ficial 2022. 6-plex IF Protocol on the BOND RX (Leica Biosystems). protocols.io https://dx.doi.org/10.17504/protocols.io.bjbzkip6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: August 04, 2020
Last Modified: July 12, 2022
Protocol Integer ID: 40025
Abstract
A 6-plex immunofluorescence protocol was optimized on Leica Bond RX Autostainer.
Preparation
Preparation

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.
Leica BOND RX Protocol
Leica BOND RX Protocol
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).