May 17, 2023

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

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.bp2l69b8dlqe/v1
  • 1Systems Immunology & Single Cell Biology group, DKFZ Heidelberg;
  • 2German Cancer Research Center (DKFZ)
Sven Truxa
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DOI: dx.doi.org/10.17504/protocols.io.bp2l69b8dlqe/v1
Protocol Citation: Sven Truxa, Felix J Hartmann 2023. MIBI: MIBI staining of fresh-frozen/OCT-embedded samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69b8dlqe/v1
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 10, 2022
Last Modified: May 17, 2023
Protocol Integer ID: 71072
Abstract
This protocol entails the recommended staining procedure for Multiplex Ion Beam Imaging Time of Flight instrument (MIBI_TOF) as developed in the Sean C. Bendall and Michael R. Angelo labs, and has been adapted in the lab of Felix Hartmann specifically for the staining of fresh-frozen samples.
CITATION
Marc Bosse, Sean Bendall, Mike Angelo. MIBI staining. protocols.io.
LINK
https://protocols.io/view/mibi-staining-b9b3r2qn


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
Target Retrieval Solution, pH 9, (3:1)Agilent (Dako)S2375
UltraPure waterInvitrogen 10977-015
Gelatin (cold water fish skin)Sigma-Aldrich G7765-250
Glutaraldehyde 8% Aqueous Solution EM GradeEMS 16020
Bovine Albumin (BSA), heat shock treatedFisher BP1600-100
Centrifugal filters (0.1µm)Millipore UFC30VV00
MIBI slidesIonPath567001
Horse serumVector LabsS-2000
EquipmentsProviderCat No.
Thermo Scientific™ Lab Vision™ PT Module Thermo Fisher Scientific A80400012
Leica ST4020 Small Linear StainerLeica14050946425
Bel-Art™ SP Scienceware™ Lab Companion Cabinet Style Vacuum Desiccators, Clear Fisher Scientific08-648-109
Seqenza staining rack + cover platesFisher Scientific73-310-017/72-110-017
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.

Initial comments
Initial comments
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
Critical
In the morning: heat up PT Module and prepare fresh antigen retrieval solution (step 6) to place in the device for preheating
Buffer preparation
Buffer preparation
20m
20m
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!

AB
ReagentsQty for 5mL
PBS 1x4750µL
Horse Serum250µL
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!


AB
ReagentsQuantity 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

Critical
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

ABC
Total volume (mL)Volume target retrieval (mL)Volume (mL) ddH2O
252.522.5
50545
1001090
Dilution table for Dako Antigen retrieval solution

Thaw and fix, wash
Thaw and fix, wash
1h
1h
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
1h
Critical
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
Antigen retrieval after fixation
Antigen retrieval after fixation
40m
40m
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.

30m
Incubation
Take slides out of the PT module and let them cool to room temperature (~10 min) before proceeding.
10m
Sequenza assembly
Sequenza assembly
Fill a disposable Pipetting Reservoir with Amount20 mL 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 Amount1 mL of wash buffer. The buffer should flow thru within 1 min 30 s. Repeat by adding 1 mL of wash buffer

5m
Blocking
Blocking
1h
1h
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 TemperatureRoom temperature for Duration01:00:00

1h
Multiplex Antibody mix
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

The total volume needed for staining each Sequenza slide assembly is Amount120 µL

Note
The manufacturer recommend to use 100 µL per Sequenza slide assembly. 120 µL is therefore a 20% excess.

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 Amount120 µL

ABCDEF
IDTargetChannelConcentration µg/mLTiter (µg/mL)Volume (µL)
1565CD45169500.250.75
1516CD8158500.51.5
..................
Total150
Antibody mix2.25
Blocking buffer147.75

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 Amount400 µL of clean blocking buffer to a Centrifugal 0.1 µm filter unit (Millipore, UFC30VV00)

Centrifigation10000 rcf, Room temperature, 00:01:00 , discard flowthrough
1m
Add antibody mix to the filter unit

Centrifigation10000 rcf, Room temperature, 00:01:00
1m
Stain 1 (Overnight)
Stain 1 (Overnight)
Add filtered antibody master mix (120µL) onto the sequenza assembly

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

10h
Post staining washing step
Post staining washing step
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 solutions
Prepare solutions
Prepare fresh 2% 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 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
Glutaraldehyde fixation
Glutaraldehyde fixation
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 Duration00:05:00

Rinse briefly with 1x PBS low barium
Dehydration and Storage
Dehydration and Storage
2h 10m
2h 10m
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

10m
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
2h