Oct 12, 2021

Public workspaceMIBI staining V.3

DOI
https://dx.doi.org/10.17504/protocols.io.byzrpx56
MIBI staining
  • Marc MB Bosse1,
  • Sean Bendall2,
  • Mike Angelo2
  • 1Stanford University;
  • 2Department of Pathology, Stanford University
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Marc Bosse
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.byzrpx56
Protocol Citation: Marc MB 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
Keywords: staining procedure, standard ffpe tissue, multiplex ion beam imaging time, mibi, multiplex ion beam imaging time of flight instrument, mibi-tof, glutaraldehyde fixation, immunohistochemistry, art of immunohistochemistry, final washes prior tissue dehydration, imaging, prior tissue dehydration, tissue
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 #567001
Troubleshooting
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).

Critical
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
Download Pt Module.Png



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.





Critical
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
Critical
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
1m
Discard flow through

Add antibody mix to the filter unit

Centrifigation10000 rcf, Room temperature, 00:01:00
1m
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 to than go to step 33

Prepare solutions
Prepare fresh glutaraldehyde fixing solution

Glutaraldehyde fixing solution

  1. Add Amount30 mL 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
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