TITLE: 6-plex IF Protocol on the BOND RX (Leica Biosystems)
AUTHORS:   Maura A Sticco-Ivins,   Abdallah Flaifel, Miriam  Ficial

[DESCRIPTION]
A 6-plex immunofluorescence protocol was optimized on Leica Bond RX Autostainer.

[STEPS]
SECTION: Preparation
1. 
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.

SECTION: Preparation
2. Bake the unstained FFPE slides in the Isotemp Oven for 30 mins at 60°C.
SECTION: Leica BOND RX Protocol
6. Bond Wash Solution (Leica Biosystems Cat# AR9590) 
Time. (min): 2:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
7. Peroxide Block (Leica Biosystems Cat# DS9800)
Time. (min): 10:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
8. Bond Wash Solution (Leica Biosystems Cat# AR9590) 
Time. (min): 4:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
9. Dako Protein Block (Agilent Cat# X090930-2)
Time. (min): 15:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
10. 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.
SECTION: Leica BOND RX Protocol
11. Bond Wash Solution (Leica Biosystems Cat# AR9590) 
Time. (min): 4:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
12. 
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



SECTION: Leica BOND RX Protocol
14. Polymer (Leica Biosystems Cat# DS9800) 
Time. (min): 10:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
15. Bond Wash Solution (Leica Biosystems Cat# AR9590) 
Time. (min): 6:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
16. Opal Fluorophore (PerkinElmer/Akoya Biosciences Cat# NEL871001KT) 
Time. (min): 5:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
17. Bond Wash Solution (Leica Biosystems Cat# AR9590) 
Time. (min): 6:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
18. Bond ER Solution 1(Leica Biosystems Cat# AR9961; citrate, pH 6.0)
Time. (min): 10:00, Temperature: 98°C, Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
19. Start a new cycle of staining from step 5 OR go to step 20



SECTION: Leica BOND RX Protocol
13. Bond Wash Solution (Leica Biosystems Cat# AR9590) 
Time. (min): 4:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
20.  Last cycle of staining:

SECTION: Leica BOND RX Protocol
20.1. Deionized Water 
Time. (min): 2:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
20.2. DAPI (PerkinElmer/Akoya Biosciences Cat# NEL871001KT)
Time. (min): 10:00; Temperature: Ambient; Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
20.3. Manually coverslip using Invitrogen ProLong Diamond Antifade Mountant (Thermo Fisher Scientific Cat# P36962). 
SECTION: Leica BOND RX Protocol
3. Deparaffinization and Rehydration 

- BOND Dewax Solution (Leica Biosystems Cat# AR9222) 
Time. (min): 30:00, Temperature: 72°C, Dispense type: open.

- Rehydration.
SECTION: Leica BOND RX Protocol
4. Bond ER Solution 1 (Leica Biosystems Cat# AR9961; citrate, pH 6.0)
Time. (min): 10:00, Temperature: 98°C, Dispense type: 150 μL.
SECTION: Leica BOND RX Protocol
5. Deionized Water 
Time. (min): 2:00; Temperature: Ambient; Dispense type: 150 μL.