Fluigent Aria 0: Sample Preparation

Catalog Numbers for Ordering Reagents:

Proteintech FlexAble 2.0 Kit and Antibodies

Ibidi Coverslips (cat# 10812)

Coating Reagents- Chrome Alum Gelatin (Newcomer Supply, cat no. 1033A USA) not available in EU or CH. *important for tissue adherence for multiplex cycles, for adherent cells can omit. Substitution Poly-D-Lysine (cat#P6407 Sigma) or Poly-L-Lysine (cat# P8920 Sigma 0.1% w/v) coating also possible

Ibidi Sticky Slides for Tissue (cat# 80518)

15mL Falcon Tubes (cat# 10773501) Thermofisher. Important to match thread size of the Aria pump.

Proteintech- Antibody Labeling with FlexAble 2.0 Kit. Use 1ug of antibody: Proteintech FlexAble calculator

Assign Antibody to Flexlinker Coralite Plus Flurophore (i.e. 488, 555, or 647).

1. Combine 1.0 ug of primary antibody (i.e, 1.4uL of GFAP 700ug/mL Cat#16825-1AY) with 2ul of FlexLinker Coralite Plus 555. Add 12.6uL of flexBuffer to bring total volume to 16uL.

2. Mix gently and icubate for 5 minutes at room temperature in the dark (in opaque microcentrifuge tubes).

3. Add 4uL of FlexQuencher.

4. Mix gently and incubate for 5 minutes at room temperature in the dark.

The Antibody is now ready to use- Store at 4 degrees.

Take Protocol of Choice: One here or steps below.

1. Perfusion with PBS followed by 4%PFA, until tissue appears white.

2. Post Fix for 1hr in 4% PFA (adjust time to tissue size).

3. Rinse 3x with PBS, and soak in Sucrose 30% overnight.

4. Fill mold with OCT, and position tissue in mold mindful of orientation for future cryosectioning.

5. Freeze in in finely crushed dry ice, wait for OCT to become opaque, before moving and storing at -80 before cyrosectioning.

Always acclimate block to cryostat temperature before sectioning.

IBEX- Recommended to coat in Chrome Alum Gelatin

Substitution Poly-D-Lysine (cat#P6407 Sigma) or Poly-L-Lysine (cat# P8920 Sigma 0.1% w/v) coating also possible

Poly D Lysine (PDL) Sigma Aldrich- P6407-5mg- substitute for original publication Chrome Alum Gelatin (Newcomer Supply, cat no. 1033A USA) not available in EU or CH. *important for tissue adherence for multiplex cycles, for adherent cells can omit.

1. Ibidi Glass Coverslips for sticky slides.Cat# 10812 #1.5H Glass Coverslip 25mm x 75 mm

2. Coat surface (marking coated side) or submerge in coating. Remove excess liquid (can be used for coating subsequent coverslips)

3. Rinse 3x with dH2O

4. Dry for at least 2 hours

5. Ready to section onto. Do not freeze once coated.

1. Section at 10 microns. Or ideal section thickness for tissue and microscope of choice acquisition.

2. Collect sections onto PDL coated (or alternative coating) Ibidi coverslips.

3. Collect extra region of interest sections on several superfrost plus slides, for validating antibody panel prior to multiplexing experiment.

4. Store at 4 degrees do not freeze until ready for mounting on Ibidi Sticky slides.

1. Prepare Blocking Buffer from 1X PBS (Gibco, cat no. 10010-023) containing 0.3% Triton-X-100 (Millipore Sigma, cat no. 93443), 1% Bovine Serum Albumin (Millipore Sigma, cat no. A1933) and

*1:100 dilution of Human (BD Biosciences, cat no. 564219) or Mouse (BD Biosciences, cat no. 553141) Fe-block or TrueStain FcX (BioLegend, cat no. 101320). • *ZMB omit the Fe in the block.

2. Prepare Antibody Dilutant from 1X PBS, with 0.1% BSA.

3. Prepare Antibodies with suggested dilutions.

4. Block test sections on Superfrost Plus slides, in humidified chamber for 20min-1hr.

5. Leave Antibody on slide for 2hrs at RT, or overnight at 4 degrees.

6. Test Exposure settings and verify that antibody dilutions are correct for your tissue of interest.

Once confirmed correct staining of single cycles from your panel on slide use same dilutions for your Fluigent Aria Multiplexing Protocol in combination with other cycles.

Example Panel for IBEX Multiplexing Experiment on Neuronal Tissue

Indirect Labeling- Primary and Secondary Antibodies can only be used in Cycle 1 in IBEX

Direct Labeled Antibodies- Conjugated Primaries can be used in all other Cycles (Cycle 2- Cycle# n) or omit indirect labeled in Cycle 1 and use conjugated for all IBEX cycles.

Load Reagents (1-8) in 15mL Falcons at correct dilutions and add freshly filtered LiBH4 and PBS to the 100mL glass bottles in reservoirs #9 and #10 respectively, label in Aria software.

Prepare the following buffers: 1X PBS, Blocking Buffer (1% BSA in 1X PBS), Antibody Dilutient (0.1%BSA in 1XPBS), Lithium Borohydrate (LiBH4_

Critical: When you prepared LiBH4 in diH20 do so in a hood and make a 1 mg/ml solution at least 5ml at a time but never more than 10mL. Leave to~incubate at room temperature for 10min, after gentle mixing. Pass the solution through a 0.22 μm syringe filter to remove any impurities.

Caution: Perform the LiBH4 preparation steps in a chemical hood with appropriate PPE. The reaction can generate hydrogen gas, which is flammable. To avoid flames, always work with small amounts (<10 mg) of Li8H4.

Store LiBH4 in a sealed container with desiccant and use a new vial of LiBH4 no later than 4 weeks after opening original container.

1. Pull away Ibidi Sticky slide film to reveal sticky coating

2. Clean excess tissue or folded sections away and make dry surface for applying sticky slide to glass coverslip

3. Align Ibidi 25mm x 75mm coverslip (cat# 10812) with Ibidi sticky slide (cat# 80518) carefully.

Do not block ports with tissue and carefully align so no over or under hangs of the glass coverslip.

Fill Chamber with PBS or TBS and avoid trapping air bubbles.

Give time for OCT from sections to dissolve and tissue to acclimate to buffer.

Tap away any bubbles in chamber before proceeding with Multiplex Experiment using Fluigent Aria Pump

1. Attach blue connector to inlet

2. Attach elbow clear connector to waste output outlet

Can manually use Fluigent Aria pump (not with software, but with Aria control panel on pump) to load fresh buffer and confirm no bubbles or leaks.

3. Feed tubing through microscope door

4. Mount sticky slide-tissue coverslip into slide carrier to load in the microscope, within the microscope.

5. Check that no tubing interferes with stage movement or acquisition.

6. Set up slide overview and acquisition settings as in this guide: Fluigent Aria 3: Combine microfluidics with ZEISS microscopes

1. Setup Aria Protocol for Multiplexing Protocol of Choice- with your Reagents.

Guide here for writing an Aria Protocol- Fluigent Aria 2: How to write an Aria Sequence

Guide here for starting the Aria Pump- Fluigent Aria 1: Startup

Guide here for Aria to Zeiss Microscope- Fluigent Aria 3: Combine microfluidics with Zeiss Microscopes

Set region of interest of your tissue section

Evaluate results from your run.

Optimize. 1. Dilutions of Antibodies 2. Placement of tissue 3. Positioning of Antibodies in Cycles. 4. Bleaching strategy (IBEX) or Elution strategy (4i) of antibodies from each cycle

Strategy to Run a Full Multiplex SeqIF

Run a real Multiplexing experiment!