MIBI: MIBI staining of fresh-frozen/OCT-embedded samples

If using frozen and vacuumed slides (e.g. from collaborators), let slides come to room temperature before destroying the vacuum seal ! (prevent condensation!) ~5-10min

In the morning: heat up PT Module and prepare fresh antigen retrieval solution (step 6) to place in the device for preheating

Prepare 1x PBS by diluting 20xPBS 1:20 in Ultrapure type 1 water (needed for blocking buffer)

Prepare Blocking buffer (also used as antibody diluent in this protocol!)

This buffer does NOT contain Tween!

A	B
Reagents	Qty for 5mL
PBS 1x	4750µL
Horse Serum	250µL

Constituents of blocking buffer

Filter with .45µm syringe

Verify stock of 1x PBS wash buffer and prepare accordingly if running low

This buffer does NOT contain Tween!

A	B
Reagents	Quantity of 250mL
PBS 10x (ml)	25mL
Bovine Albumin (BSA), heat shock treated (g)	0.25g
Ultrapure (type 1) water (mL)	225mL

Constituents of PBS washing buffer

To prepare fresh antigen retrieval solution , dilute 10x DAKO (3-in-1) pH9 antigen retrieval solution 1:10 in ultrapure (type 1) water (e.g. for 25mL: 2.5mL + 22.5mL)

note: if retrieving in gold slide delivery chamber (5 slots), 25mL are sufficient

A	B	C
Total volume (mL)	Volume target retrieval (mL)	Volume (mL) ddH2O
25	2.5	22.5
50	5	45
100	10	90

Dilution table for Dako Antigen retrieval solution

If embedding medium is still on slide, carefully dip slide into 1x PBS before fixation

Fix Slides after thawing in 10% Neutral Buffered Formaline (NBF) for 1h at room temperature

Transfer the slides in the first MIBI 1x PBS wash buffer and dip shortly to remove PFA

Transfer the slides to the second 1x PBS wash buffer and dip shortly to remove PFA

After washing, transfer slide into antigen retrieval buffer jar within the preheated PT module

press run on the digital screen as soon as the device is preheated to 75°C. The display will show "WARMUP" and heat up to 80°C for retrieval , stay at this temperature for 20minutes, direcly put tissue back to RT.This process takes ~30min.

Take slides out of the PT module and let them cool to room temperature (~10 min) before proceeding.

Fill a disposable Pipetting Reservoir with 20mL of 1x PBS wash buffer

Place sample slide on a Sequenza cover plate aligning the bottom slide with the notches on the cover plate (see picture below panel A)

Fill by capillarity the space between the slide and cover plate by holding the parts tight and dipping the bottom part of the assembly in the wash buffer reservoir (see picture above panel B).

If capillary force is not sufficient to fill the staining reservoir, a careful pumping motion on the triangular protrusion can help.

Transfer the slide and coverplate assembly into the Sequenza rack. Slip in the assembly (see below picture panel C)

Secure the assembly and make sure that the assembly placed down in the rack (see above picture panel D)

Add 1mL of wash buffer. The buffer should flow thru within 1 min 30 s. Repeat by adding 1 mL of wash buffer

Add 200µL of blocking buffer to the sequenza assembly, make sure the buffer is retained in the assembly (the waterline should stop at the upper end of the capillary space)

Incubate at Room temperature for 1h 0m 0s

Prepare antibody mix based on the putative multiplex antibody panel

Make sure that all the antibodies are ready to use BEFORE starting to build the panel

It is highly recommended to prepare all the antibodies, ready to use, a day before the panel is built

The total volume needed for staining each Sequenza slide assembly is 120µL

Build an antibody mix table information to make the antibody panel as follow:

Conjugation ID, Target name, Channel, Antibody concentration, Titer, Volume

Exemple:

The total volume needed for staining each Sequenza slide assembly is 120µL

A	B	C	D	E	F
ID	Target	Channel	Concentration µg/mL	Titer (µg/mL)	Volume (µL)
1565	CD45	169	50	0.25	=5*E2/D2
1516	CD8	158	50	0.5	=5*E3/D3
...	...	...	...	...	...
				Total	150
				Antibody mix	=F5-F7
				Blocking buffer	=F5-SUM(F2,F3)

For Blocking Buffer solution preparation refer to step 4

Prepare the antibody mix according to the calculation. Make sure to spin down antibody vials before pipetting to prevent pipetting of aggregates. Use filter tips when pipetting from communal stocks and keep the tubes on ice.

Pre-wet spin column

Add 400µL of clean blocking buffer to a Centrifugal 0.1 µm filter unit (Millipore, UFC30VV00)

10000rcf , discard flowthrough

Add antibody mix to the filter unit

10000rcf

Add filtered antibody master mix (120µL) onto the sequenza assembly

Place the moist chamber at 4˚C , preferably in a place with low disturbance

Following overnight incubation, wash twice with 1 mL of wash buffer

After the 1h incubation, wash twice with PBS wash buffer within the sequenza assembly by pipetting 2x 1mL.

Prepare fresh 2% glutaraldehyde fixing solution

Glutaraldehyde fixing solution

1. Add 30mL of 1x PBS low barium in a 50 mL tube
2. Break the glass glutaraldehyde 8% (amber vial)
3. Add the glutaraldehyde (10 mL) to the diluent by inverting it and tapping the bottom of the vial
4. Transfer the content in a linear stainer container

Set the linear stainer containers

Fill containers with the following solution and order

Glutaraldehyde x 1, PBS low barium x 1, TRIS 100 mm pH 8.5 x 3, ddH2O x 2, 70% Ethanol x1, 80% Ethanol x1, 95% Ethanol x 2, 100% Ethanol x 2, exit stainless steel tank = empty

Disassemble the sequenza setup. Make sure at this point that the slide doesn't dry out and directly proceed to the next steps.

Mount the slides on the linear slide holder

Fix in 2% glutaraldehyde for 0h 5m 0s

Rinse briefly with 1x PBS low barium

Press on Menu

Check for Processing time = 30 sec, Lift bar = 976, Number of dips = 3

Continue to press Menu until the screen displays Start at: __

Set Start position corresponding to the first slide carrier position

Exemple: If the first slide carrier is at position 3, use Plus ( + ) or Minus ( - ) button to increase or decrease to get Start at: 03

Then press Enter

Press Run on the Linear Stainer

Allow the dehydration process and wait until the slides reached the empty stainless steel tank and stop

Store the slides immediately under vacuum until MIBI acquisition

Alternatively, the stained slides can be stored in a vacuum sealed bag for longterm storage pre and post MIBI acquisition