MIBI staining

FFPE or frozen sections should be deposited on special conductive slides for MIBI

It is recommended to use freshly cut tissue sections. Otherwise tissue section slides should be stored properly using different state of the art methods (vacuum chamber, nitrogen chamber or vacuum sealed bags)

Bake the sections at 70°C for 0h 20m 0s in a dry incubator

 **Optional**  : `1h 0m 0s` ; `1h 0m 0s`

Last 10 min place the slide (s) vertically with the label side up to allow drip down the paraffin

Prepare Target retrieval solution

2.5mL of target retrieval solution 10x (3-in-1), DAKO

in 22.5mL of ultrapure (type 1, >18 MOhms) water

A	B	C
Total volume (mL)	Volume (mL) Target retrieval	Volume (mL) H2O
25	2.5	22.5
50	5	45
100	10	90

Put in the containers with the diluted target retrieval solution in the PT Module

Equipment

Value	Label
Thermo ScientificTM Lab VisionTM PT Module	NAME
Programmed Water bath	TYPE
Thermofisher	BRAND
A80400012	SKU

PT Module Preheat

Press RUN on digital screen and check for PREHEAT 75 on display

Linear Stainer

Pour out reagent containers and fill with fresh reagents:

Xylene x 3, 100% Ethanol x 2, 95% Ethanol x 2, 80% Ethanol, 70% Ethanol, ddH2O x 2, exit stainless steel tank = ultrapure (type 1, >18 MOhms) water

Insert slides into slide carriers

Place the slide carrier into first xylene container

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 4, use Plus ( + ) or Minus ( -) button to increase or decrease to get Start at: 04

Then press Enter

Synchronize when the PT module temperature has reached 75˚C

then Press Run on the Linear Stainer

Allow the rehydration process and wait until the slides have reached the stainless steel tank and stop

Bring the stainless steel tank with the slides in close to the PT module

Open the PT Module and insert the slides in the warm Target retrieval solution container

Discard water immediately from the stainless steel tank

Press RUN again and check for first WARMUP then HEAT on display, once the temperature has reached 97˚C

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

A	B
Reagents	Qty for 1000 mL
PBS IHC Wash Buffer with Tween 20 (mL)	50
Bovine Albumin (BSA), heat shock treated (g)	1
Ultrapure (type 1) water (mL)	949

Allow to run for 40 min at97°C and then cool down for approximately 50 min and reach 65°C

When the alarm sounds Stop the PT module

Take out the slides and let cool down at room temperature for at least 0h 5m 0s

Prepare two coplin jars filled with MIBI 1x PBS wash buffer

Transfer the slides in the first MIBI 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm

Transfer the slides to the second 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm

Make sure to dry with a folded-tissue paper the slide, leaving a square of wet surface surrounding of the tissue section

Note: Do not let AIR DRY the tissue section, this will result high background and false positive staining

Draw a square following the outside edges of the wet square with an hydrophobic barrier pen (ImmEdge pen)

If a biotinylated antibody or a probe is used, it is recommended to block endogenous biotin

Place the slides in the moist chamber

Add drops of Avidin solution (Avidin/Biotin blocking kit, Biolegend) sufficient to cover the sample and incubate for 0h 10m 0s at Room temperature

Wash in coplin jar with MIBI 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm

Add drops of Biotin solution sufficient to cover the sample and incubate for 0h 10m 0s at Room temperature

Wash in coplin jar with MIBI 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm

Next day, use anti-biotin metal-labeled antibody (clone 1D4-C5) in Stain 2 panel

Add 100 µL of Blocking Buffer for 18 mm²

For blocking solution preparation refer to MIBI and IHC solutions protocols

A	B	C	D	E	F
Estimated Surface area (mm)	10x10	15x15	18x18	20x20	20x45
Volume (µL)	50	70	100	150	350

Place the slides in a moist chamber at Room temperature and incubate 0h 20m 0s to 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

Evaluate the total volume of multiplex antibody mix by counting the number of slides and the surface area per slide

Refer to the chart for the volume of antibody to apply according to the estimated surface area

A	B	C	D	E	F
Estimated Surface area (mm)	10x10	15x15	18x18	20x20	20x45
Volume (µL)	50	70	100	150	350

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

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

Exemple:

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	500
				Antibody mix	=F5-F7
				Antibody diluent (NHS 3%)	=F5-SUM(F2,F3)

For Antibody Diluent (NHS 3%) solution preparation refer to MIBI and IHC solutions protocols

Add 400µL of antibody diluent (NHS 3%) to a Centrifugal 0.1 µm filter unit (Millipore, UFC30VV00)

10000rcf

Discard flow through

Add antibody mix to the filter unit

10000rcf

Remove the blocking solution by tapping the slide on a side

Immediately add the filtered multiplex antibody mix

Place the moist chamber at 4˚C , preferably in a place with low disturbance (e.g. a designated area in a cold room)

Prepare two Coplin jars filled with 1x PBS wash buffer

Transfer the slides into the first Coplin jar and use orbital shaker set for 5 min, 70 rpm

Transfer the slides into the second Coplin jar and use orbital shaker set for 5 min, 70 rpm

Add adequate volume of the selected sub-panel of antibody mix

Refer to the chart for the volume of antibody to apply

A	B	C	D	E	F
Estimated Surface area (mm)	10x10	15x15	18x18	20x20	20x45
Volume (µL)	50	70	100	150	350
Estimated # of drops	1	2	3	4	8-9

Place sample in a sealed humidity chamber, transfer to 4^oC refrigerator, and incubate 1h 0m 0s

After 1h incubation

than go to step 33

Prepare fresh 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 content of the glutaraldehyde (10 mL) by inverting it and tapping the bottom of the vial in the 50 mL tube
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

Mount the slides on the linear slide holder

Fix 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