TITLE: MIBI staining
AUTHORS: Marc Bosse, Sean Bendall, Mike Angelo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.   </div></div>

[STEPS]
?. [Slide for MIBI]
FFPE or frozen sections should be deposited on special conductive slides for MIBIIt 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)
?. [Slide baking and PT module preparation ]
Bake the sections at  for  in a dry incubator    Optional :  ;
70 °C
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).
?. [Slide baking and PT module preparation ]
Last 10 min place the slide (s) vertically with the label side up to allow drip down the paraffin
?. [Slide baking and PT module preparation ]
Prepare Target retrieval solution  of target retrieval solution 10x (3-in-1), DAKOin  of ultrapure (type 1,  >18 MOhms) water   ABC1Total volume (mL)Volume (mL) Target retrievalVolume (mL) H2O2252.522.535054541001090
2.5 mL
22.5 mL
ABC1Total volume (mL)Volume (mL) Target retrievalVolume (mL) H2O2252.522.535054541001090
?. [Slide baking and PT module preparation ]
Put in the containers with the diluted target retrieval solution in the PT Module
?. [Slide baking and PT module preparation ]
PT Module Preheat Press RUN on digital screen and check for PREHEAT 75 on display
?. [Slide deparafination]
Linear StainerPour 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
IMPORTANT: Use fresh xylene for every deparafination.
?. [Slide deparafination]
Insert slides into slide carriersPlace the slide carrier into first xylene container
?. [Slide deparafination]
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
?. [Slide deparafination]
Then press Enter
?. [Slide deparafination]
Synchronize when the PT module temperature has reached 75˚C then Press Run on the Linear Stainer
?. [Slide deparafination]
Allow the rehydration process and wait until the slides have reached the stainless steel tank and stop
?. [Slide deparafination]
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 containerDiscard water immediately from the stainless steel tank
?. [Antigen Retrieval]
Press RUN again and check for first  WARMUP  then  HEAT on display, once the temperature has reached 97˚C
?. [Antigen Retrieval]
Verify stock of 1x PBS wash buffer and prepare accordingly if running low  AB1ReagentsQty for  1000 mL2PBS IHC Wash Buffer with Tween 20 (mL)503Bovine Albumin (BSA), heat shock treated (g)14Ultrapure (type 1) water (mL)949
AB1ReagentsQty for  1000 mL2PBS IHC Wash Buffer with Tween 20 (mL)503Bovine Albumin (BSA), heat shock treated (g)14Ultrapure (type 1) water (mL)949
?. [Antigen Retrieval]
Allow to run for 40 min at and then cool down for approximately 50 min and reach
97 °C
65 °C
?. [Antigen Retrieval]
When the alarm sounds Stop the PT module
?. [Antigen Retrieval]
Take out the slides and let cool down at room temperature for at least
?. [Antigen Retrieval]
Prepare two coplin jars filled with MIBI 1x PBS wash buffer
?. [Antigen Retrieval]
Transfer the slides in the first MIBI 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm
?. [Antigen Retrieval]
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
?. [Hydrophobic barrier pen]
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 biotinPlace the slides in the moist chamberAdd drops of Avidin solution (Avidin/Biotin blocking kit, Biolegend) sufficient to cover the sample and incubate for  at
0 Room temperature
?. [Optionnal: blocking endegenous biotin ]
Wash in coplin jar with MIBI 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm
?. [Optionnal: blocking endegenous biotin ]
Add drops of Biotin solution sufficient to cover the sample and incubate for  at
0 Room temperature
?. [Optionnal: blocking endegenous biotin ]
Wash in coplin jar with MIBI 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm
?. [Optionnal: blocking endegenous biotin ]
Next day, use anti-biotin metal-labeled antibody (clone 1D4-C5) in Stain 2 panel
?. [Blocking ]
Add 100 µL of Blocking Buffer for 18 mm2For blocking solution preparation refer to MIBI and IHC solutions protocols  ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
?. [Blocking ]
Place the slides in a moist chamber at  and incubate   to
0 Room temperature
?. [Multiplex Antibody mix]
Prepare antibody mix based on the putative multiplex antibody panelMake sure that all the antibodies are ready to use BEFORE starting to build the panelIt is highly recommended to prepare all the antibodies, ready to use, a day before the panel is built
?. [Multiplex Antibody mix]
Evaluate the total volume of multiplex antibody mix by counting the number of slides and the surface area per slideRefer to the chart for the volume of antibody to apply according to the estimated surface area  ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
?. [Multiplex Antibody mix]
Build an antibody mix table information to make the antibody panel as follow:Conjugation ID, Target name, Channel, Antibody concentration, Titer, Volume Exemple:  ABCDEF1IDTargetChannelConcentration µg/mLTiter (µg/mL)Volume (µL)21565CD45169500.25=$F$5*E2/D231516CD8158500.5=$F$5*E3/D34..................5Total5006Antibody mix=F5-F77Antibody diluent (NHS 3%)=F5-SUM(F2,F3)For Antibody Diluent (NHS 3%)  solution preparation refer to MIBI and IHC solutions protocols
ABCDEF1IDTargetChannelConcentration µg/mLTiter (µg/mL)Volume (µL)21565CD45169500.25=$F$5*E2/D231516CD8158500.5=$F$5*E3/D34..................5Total5006Antibody mix=F5-F77Antibody diluent (NHS 3%)=F5-SUM(F2,F3)
?. [Multiplex Antibody mix]
Add  of antibody diluent (NHS 3%) to a Centrifugal 0.1 µm filter unit (Millipore, UFC30VV00)
400 µl
?. [Multiplex Antibody mix]
Centrifuge: 10000 1324, 1 min
?. [Multiplex Antibody mix]
Discard flow through
?. [Multiplex Antibody mix]
Add antibody mix to the filter unit
?. [Multiplex Antibody mix]
Centrifuge: 10000 1324, 1 min
?. [Stain 1 (Overnight)]
Remove the blocking solution by tapping the slide on a sideImmediately add the filtered multiplex antibody mix
?. [Stain 1 (Overnight)]
Place the moist chamber at 4˚C , 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
?. [Wash buffer]
Transfer the slides into the first Coplin jar and use orbital shaker set for 5 min, 70 rpm
?. [Wash buffer]
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 mixRefer to the chart for the volume of antibody to apply  ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)50701001503503Estimated # of drops12348-9
ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)50701001503503Estimated # of drops12348-9
?. [Stain 2 (1h)]
Place sample in a sealed humidity chamber, transfer to 4oC refrigerator, and incubate
?. [Wash buffer]
After 1h incubation   than go to step 32
?. [Prepare solutions]
Prepare fresh glutaraldehyde fixing solutionGlutaraldehyde fixing solutionAdd  of 1x PBS low barium in a 50 mL tubeBreak the glass glutaraldehyde 16% (amber vial) Add the content of the glutaraldehyde (10 mL) by inverting it and tapping the bottom of the vial in the 50 mL tubeTransfer the content in a linear stainer container
30 mL
?. [Prepare solutions]
Set the linear stainer containersFill containers with the following solution and orderGlutaraldehyde 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 holderFix for
?. [Glutaraldehyde fixation  ]
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
?. [Dehydration and Storage]
Then press Enter
?. [Dehydration and Storage]
Press Run on the Linear Stainer
?. [Dehydration and Storage]
Allow the dehydration process and wait until the slides reached the empty stainless steel tank and stop
?. [Dehydration and Storage]
Store the slides immediately under vacuum until MIBI acquisitionAlternatively, the stained slides can be stored in a vacuum sealed bag for longterm storage pre and post MIBI acquisition