MIBI staining

Marc MB Bosse; Sean Bendall; Mike Angelo

Oct 12, 2021

Version 3

MIBI staining V.3

DOI

dx.doi.org/10.17504/protocols.io.byzrpx56

Marc Bosse¹,
Sean Bendall²,
Mike Angelo²

¹Stanford University;
²Department of Pathology, Stanford University

Marc Bosse

Stanford University

DOI: dx.doi.org/10.17504/protocols.io.byzrpx56

Protocol Citation: Marc Bosse, Sean Bendall, Mike Angelo 2021. MIBI staining. protocols.io https://dx.doi.org/10.17504/protocols.io.byzrpx56Version created by Marc Bosse

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: October 12, 2021

Last Modified: October 12, 2021

Protocol Integer ID: 54033

Abstract

This protocol is the standard FFPE tissue staining procedure recommended for Multiplex Ion Beam Imaging Time of Flight instrument (MIBI_TOF) developed in the Sean C. Bendall and Michael R. Angelo labs. The protocol has been successfully used for MIBI and is the result of extensive optimization experiments. It is inspired from state-of-the art of immunohistochemistry staining procedures but differs in some very important steps, namely, glutaraldehyde fixation and final washes prior tissue dehydration. Failure to follow exactly all steps described in this procedure may result in inconsistencies in output data after MIBI_TOF acquisition.   

Guidelines

Staining tissue sections is fairly straightforward but there are few things to be cognizant of, when preparing samples:
Always try to limit mechanical damage to the sample surface. This can occur when moving the samples with forceps, particularly when the mounting substrates are small.
Once the samples have been rehydrated, they cannot dry out until the end of the protocol.
Be careful at all times to not touch the tissue, in order to not leave any residue.
Once the samples have been stained, fixed, and dehydrated, they have an indefinite shelf life and can be imaged at any time but need to be stored properly such as in a vacuum chamber or in a sealed vacuum bag
MIBI_TOF observes the basic principles of Mass spectrometry. Any contaminant ions present in water or in the air can potentially compromise the integrity of the sample.
Therefore to prevent potential contamination, it is important to always use single use lab ware containers to make the solutions. The protocol has been validated using the level of precision of graduated Nalgene bottles and 50 mL tubes. 
The use of washed beakers or graduated cylinders is not considered as good a practice, due possible introduction of contaminants (exemples of sources: barium from lab ware soap or calcium from air dried lab ware)

Materials

 
 
ABC
ProductsProviderCatalogue No.
Alcohol ethyl ETHANOL 200 PROOF  Gold Shield  412811  
Alcohol ethyl ETHANOL 190 PROOF  Gold Shield  412602  
TBS IHC Wash Buffer with Tween 20Cell Marque  935B-09  
PBS IHC Wash Buffer with Tween 20Cell Marque934B-09  
Target Retrieval Solution, pH 9, (3:1)Agilent (Dako)S2375  
UltraPure waterInvitrogen  10977-015  
Avidin/Biotin Blocking KitBiolegend  927301  
Hydrogen peroxideSigma216763-100ML
Gelatin (cold water fish skin)Sigma-Aldrich  G7765-250
Xylene HISTOLOGICAL GRADESigma-Aldrich   534056-500  
Glutaraldehyde 8% Aqueous Solution EM GradeEMS   16020  
Bovine Albumin (BSA), heat shock treatedFisher  BP1600-100
Centrifugal filters (0.1µm)Millipore    UFC30VV00  
ImmEdge hybrophobic barrier penVector labH-4000
MIBI slidesIonPath567001
LevamisoleVector LabsSP-5000
Horse serumVector LabsS-2000
VectaMount Permanent Mounting MediumVector LabsH-5000
EquipmentsProviderCat No.
Thermo Scientific™ Lab Vision™ PT Module  Thermo Fisher Scientific A80400012  
Leica ST4020 Small Linear StainerLeica14050946425  
Digital incubators, INCU-Line®, IL 10 and IL 23  VWR390-0384  
Bel-Art™ SP Scienceware™ Lab Companion Cabinet
  Style Vacuum Desiccators, Clear  Fisher Scientific08-648-109  
Oribital shakerBoekel 270200  
Moist chamberTed Pella21051
  

Protocol materials

ReagentMIBI slidesIonPathCatalog #567001Step 1
 

Safety warnings

All organic solvents should be manipulated under a chemical hood.

Before start

Verify the stocks of all reagents and place an order or prepare solutions, if some reagents are running low.

Slide for MIBI

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)
ReagentMIBI slidesIonPathCatalog #567001  

Slide baking and PT module preparation

Bake the sections at Temperature70 °C   for Duration00:20:00   in a dry incubator
    Optional : Duration01:00:00   ; DurationOvernight  
   
Note
Note: Some tissues or section size may need longer baking time. 

Recommended to bake at least 1 hour for brain tissue or TMA. This can be extended to 16 h (overnight).

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

Prepare Target retrieval solution
Amount2.5 mL    of target retrieval solution 10x (3-in-1), DAKO
in Amount22.5 mL   of ultrapure (type 1,  >18 MOhms) water 

Total volume (mL)Volume (mL) Target retrievalVolume (mL) H2O
252.522.5
50545
1001090

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


 

Equipment
Thermo ScientificTM Lab VisionTM PT Module
NAME
Programmed Water bath
TYPE
Thermofisher
BRAND
A80400012
SKU
https://www.thermofisher.com/order/catalog/product/A80400012#/A80400012
LINK

PT Module Preheat 
Press RUN on digital screen and check for PREHEAT 75 on display

Slide deparafination

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 


Note
IMPORTANT: Use fresh xylene for every deparafination.

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

Antigen Retrieval

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


ReagentsQty 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 atTemperature97 °C   and then cool down for approximately 50 min and reach Temperature65 °C  

When the alarm sounds Stop the PT module

Take out the slides and let cool down at room temperature for at least Duration00:05:00   

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

Hydrophobic barrier pen

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)

Optionnal: blocking endegenous biotin

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 Duration00:10:00   at TemperatureRoom 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 Duration00:10:00   at TemperatureRoom 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

Blocking

Add 100 µL of Blocking Buffer for 18 mm2

For blocking solution preparation refer to MIBI and IHC solutions protocols

Estimated Surface area (mm)10x1015x1518x1820x2020x45
Volume (µL)5070100150350

Place the slides in a moist chamber at TemperatureRoom temperature   and incubate  Duration00:20:00   to  Duration01:00:00   

Multiplex Antibody mix

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
Estimated Surface area (mm)10x1015x1518x1820x2020x45
Volume (µL)5070100150350

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


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

Exemple:
ABCDEF
IDTargetChannelConcentration µg/mLTiter (µg/mL)Volume (µL)
1565CD45169500.252.5
1516CD8158500.55
..................
Total500
Antibody mix7.5
Antibody diluent (NHS 3%)492.5

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

Add Amount400 µL   of antibody diluent (NHS 3%) to a Centrifugal 0.1 µm filter unit (Millipore, UFC30VV00)

Centrifigation10000 rcf, Room temperature, 00:01:00  

Discard flow through

Add antibody mix to the filter unit 

Centrifigation10000 rcf, Room temperature, 00:01:00  

Stain 1 (Overnight)

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 DurationOvernight  , preferably in a place with low disturbance (e.g. a designated area in a cold room)

Wash buffer

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

Stain 2 (1h)

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

Refer to the chart for the volume of antibody to apply

Estimated Surface area (mm)10x1015x1518x1820x2020x45
Volume (µL)5070100150350
Estimated # of drops12348-9

Place sample in a sealed humidity chamber, transfer to 4oC refrigerator, and incubate Duration01:00:00   

Wash buffer

After 1h incubation 
Go togo to step #29    than go to step 33

Prepare solutions

Prepare fresh glutaraldehyde fixing solution

Glutaraldehyde fixing solution

Add Amount30 mL   of 1x PBS low barium in a 50 mL tube
Break the glass glutaraldehyde 8% (amber vial) 
Add the content of the glutaraldehyde (10 mL) by inverting it and tapping the bottom of the vial in the 50 mL tube
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

Glutaraldehyde fixation

Mount the slides on the linear slide holder

Fix for Duration00:05:00   

Rinse briefly with 1x PBS low barium 

Dehydration and Storage

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

A	B	C
Products	Provider	Catalogue No.
Alcohol ethyl ETHANOL 200 PROOF	Gold Shield	412811
Alcohol ethyl ETHANOL 190 PROOF	Gold Shield	412602
TBS IHC Wash Buffer with Tween 20	Cell Marque	935B-09
PBS IHC Wash Buffer with Tween 20	Cell Marque	934B-09
Target Retrieval Solution, pH 9, (3:1)	Agilent (Dako)	S2375
UltraPure water	Invitrogen	10977-015
Avidin/Biotin Blocking Kit	Biolegend	927301
Hydrogen peroxide	Sigma	216763-100ML
Gelatin (cold water fish skin)	Sigma-Aldrich	G7765-250
Xylene HISTOLOGICAL GRADE	Sigma-Aldrich	534056-500
Glutaraldehyde 8% Aqueous Solution EM Grade	EMS	16020
Bovine Albumin (BSA), heat shock treated	Fisher	BP1600-100
Centrifugal filters (0.1µm)	Millipore	UFC30VV00
ImmEdge hybrophobic barrier pen	Vector lab	H-4000
MIBI slides	IonPath	567001
Levamisole	Vector Labs	SP-5000
Horse serum	Vector Labs	S-2000
VectaMount Permanent Mounting Medium	Vector Labs	H-5000
Equipments	Provider	Cat No.
Thermo Scientific™ Lab Vision™ PT Module	Thermo Fisher Scientific	A80400012
Leica ST4020 Small Linear Stainer	Leica	14050946425
Digital incubators, INCU-Line®, IL 10 and IL 23	VWR	390-0384
Bel-Art™ SP Scienceware™ Lab Companion Cabinet Style Vacuum Desiccators, Clear	Fisher Scientific	08-648-109
Oribital shaker	Boekel	270200
Moist chamber	Ted Pella	21051

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

	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

	Estimated Surface area (mm)	10x10	15x15	18x18	20x20	20x45
	Volume (µL)	50	70	100	150	350

A	B	C	D	E	F
ID	Target	Channel	Concentration µg/mL	Titer (µg/mL)	Volume (µL)
1565	CD45	169	50	0.25	2.5
1516	CD8	158	50	0.5	5
...	...	...	...	...	...
				Total	500
				Antibody mix	7.5
				Antibody diluent (NHS 3%)	492.5

Public workspaceMIBI staining V.3

MIBI staining V.3