TITLE: MIBI: MIBI staining of fresh-frozen/OCT-embedded samples
AUTHORS: Sven Truxa, Felix J Hartmann

[DESCRIPTION]
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.
 



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


[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)

[STEPS]
SECTION: Buffer preparation
5. Verify stock of 1x PBS wash buffer and prepare accordingly if running low
This buffer does NOT contain Tween! 


 

SECTION: Thaw and fix, wash
9. Transfer the slides in the first MIBI 1x PBS wash buffer and dip shortly to remove PFA

SECTION: Thaw and fix, wash
10. Transfer the slides to the second 1x PBS wash buffer and dip shortly to remove PFA
SECTION: Blocking
19. 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)

SECTION: Blocking
20. Incubate at Room temperature for 60 min 

SECTION: Multiplex Antibody mix
21. 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

SECTION: Multiplex Antibody mix
21.1. The total volume needed for staining each Sequenza slide assembly is 120 µL

 

SECTION: Multiplex Antibody mix
21.2. 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 120 µL

 

 
For Blocking Buffer solution preparation refer to step 4





SECTION: Multiplex Antibody mix
22. Pre-wet spin column

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

SECTION: Multiplex Antibody mix
22.1. 10000 rcf, 1 min , discard flowthrough
SECTION: Multiplex Antibody mix
23. Add antibody mix to the filter unit 

SECTION: Multiplex Antibody mix
23.1. 10000 rcf, 1 min 
SECTION: Stain 1 (Overnight)
24. Add filtered antibody master mix (120µL) onto the sequenza assembly

SECTION: Stain 1 (Overnight)
25. Place the moist chamber at 4˚C  , preferably in a place with low disturbance

SECTION: Post staining washing step
26. Following overnight incubation, wash twice with 1 mL of wash buffer
27. After the 1h incubation, wash twice with PBS wash buffer within the sequenza assembly by pipetting 2x 1mL. 

SECTION: Prepare solutions
28. Prepare fresh 2% glutaraldehyde fixing solution

Glutaraldehyde fixing solution

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

SECTION: Prepare solutions
29. 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
SECTION: Glutaraldehyde fixation
31. Mount the slides on the linear slide holder

Fix in 2% glutaraldehyde for 5 min 

SECTION: Glutaraldehyde fixation
32. Rinse briefly with 1x PBS low barium 
SECTION: Dehydration and Storage
33. 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 

SECTION: Dehydration and Storage
33.1. Then press Enter 
SECTION: Dehydration and Storage
33.2. Press Run on the Linear Stainer
SECTION: Dehydration and Storage
33.3. Allow the dehydration process and wait until the slides reached the empty stainless steel tank and stop
SECTION: Dehydration and Storage
33.4. 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
SECTION: Initial comments
1. 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
SECTION: Buffer preparation
4. Prepare Blocking buffer (also used as antibody diluent in this protocol!)
This buffer does NOT contain Tween!

 

Filter with .45µm syringe
SECTION: Buffer preparation
3. Prepare 1x PBS by diluting 20xPBS 1:20 in Ultrapure type 1 water (needed for blocking buffer)
SECTION: Thaw and fix, wash
8. Fix Slides after thawing in 10% Neutral Buffered Formaline (NBF) for 1h at room temperature
SECTION: Antigen retrieval after fixation
11. 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.

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

 

SECTION: Antigen retrieval after fixation
12. Take slides out of the PT module and let them cool to room temperature (~10 min) before proceeding. 
SECTION: Sequenza assembly
13. Fill a disposable Pipetting Reservoir with 20 mL of 1x PBS wash buffer
SECTION: Sequenza assembly
14. Place sample slide on a Sequenza cover plate aligning the bottom slide with the notches on the cover plate (see picture below panel A)

  

SECTION: Sequenza assembly
15. 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.
SECTION: Sequenza assembly
16. Transfer the slide and coverplate assembly into the Sequenza rack. Slip in the assembly (see below picture panel C)

  
SECTION: Sequenza assembly
17. Secure the assembly and make sure that the assembly placed down in the rack (see above picture panel D)

SECTION: Sequenza assembly
18. Add 1 mL of wash buffer. The buffer should flow thru within 1 min 30 s. Repeat by adding 1 mL of wash buffer

SECTION: Multiplex Antibody mix
21.3. 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.
SECTION: Glutaraldehyde fixation
30. Disassemble the sequenza setup. Make sure at this point that the slide doesn't dry out and directly proceed to the next steps.
SECTION: Thaw and fix, wash
7. If embedding medium is still on slide, carefully dip slide into 1x PBS before fixation