TITLE: Staining Sequenza
AUTHORS: Marc Bosse, Sean Bendall, Mike Angelo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the use of sequenza staining device. The sequenza can be use for MIBI or IHC staining methods.</div></div>

[STEPS]
?. [Sequenza assembly]
Fill a disposable Pipetting Reservoir with  of 1x PBS wash buffer
20 mL
?. [Sequenza assembly]
Place sample slide on a Sequenza cover plate aligning the bottom slide with the notches on the cover plate (see picture below panel A)
?. [Sequenza assembly]
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)
?. [Sequenza assembly]
Transfer the slide and coverplate assembly into the Sequenza rack. Slip in the assembly (see below picture panel C)
?. [Sequenza assembly]
Secure the assembly and make sure that the assembly placed down in the rack (see above picture panel D)
?. [Blocking]
Add  of Blocking buffer)
200 µl
?. [Blocking]
Place the cover and incubate with the blocking buffer for 1h
?. [Sequenza assembly]
Add  of wash buffer. The buffer should flow thru within 1 min 30 s. Repeat by adding 1 mL of wash buffer
1 mL
?. [Optional: blocking endogeous biotin]
If you are using biotinylated antibody or probe, you need to block endogenous biotinAdd 3 drops of Avidin solution sufficient to cover the sample and incubate for
0 Room temperature
?. [Optional: blocking endogeous biotin]
Add 1 mL of wash buffer
?. [Optional: blocking endogeous biotin]
Add drops Biotin solution sufficient to cover the sample and incubate for
0 Room temperature
?. [Optional: blocking endogeous biotin]
Add  of wash buffer
1 mL
?. [Optional: blocking endogeous biotin]
Next day, use anti-biotin metal-labeled antibody (1D4-C5) in Stain 2 panel
?. [Optional: blocking endogeous biotin]
If presence of bubbles are observed after adding Avidin and Biotin solution.Dismount the slide and the Sequenza cover plate
?. [Optional: blocking endogeous biotin]
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)
?. [Prepare antibody master mix]
Spin down briefly all antibody tubes at  for , preferably at
Centrifuge: 10.000 34
4 °C
?. [Prepare antibody master mix]
Pipet the respective amount of each individual antibody. Do not disturb the bottom of the antibody tube when pipetting
?. [Prepare antibody master mix]
The total volume needed for staining each Sequenza slide assembly is
120 µl
The manufacturer recommend to use 100 µL per Sequenza slide assembly. 120 µL is therefore a 20% excess.
?. [Prepare antibody master mix]
Prepare 0.1 µm filter unit
?. [Prepare antibody master mix]
Prepare a panel table in excel including the following information: Conjugation ID, Target name, Channel, , Antibody concentration, Titer, Volume Exemple:  ABCDEF1IDTarget nameChannelConcentration (ug/mL)Titer  (ug/mL)Calculation formulation for Volume (µL)21299CD4143501=$F$6*E2/D231221CD11c161500.25=$F$6*E3/D341292CD3159500.5=$F$6*E4/D45..................6Total volume (µL)1507Antibody diluent volume=F6-SUM(F2,F3,F4)8Antibody mix volume=F6-F7
ABCDEF1IDTarget nameChannelConcentration (ug/mL)Titer  (ug/mL)Calculation formulation for Volume (µL)21299CD4143501=$F$6*E2/D231221CD11c161500.25=$F$6*E3/D341292CD3159500.5=$F$6*E4/D45..................6Total volume (µL)1507Antibody diluent volume=F6-SUM(F2,F3,F4)8Antibody mix volume=F6-F7
?. [Prepare antibody master mix]
Add first, antibody diluent (NHS 3%) to complete to the total volume
?. [Prepare antibody master mix]
Add 400 µL of antibody diluent buffer (NHS 3%) to a 0.1 µm centrifugal filter device
?. [Prepare antibody master mix]
Spin at 10,000 x g for 1 min
?. [Prepare antibody master mix]
Discard the flowthough by aspirating
?. [Prepare antibody master mix]
Add the antibody mix to the filter unit
?. [Prepare antibody master mix]
Spin at 10,000 x g for 1 min
?. [Stain 1 (overnight)]
After, blocking incubation, add  of antibody buffer (NHS 3%)
200 µl
?. [Stain 1 (overnight)]
Make sure that the antibody buffer has passed through to prevent dilution of antibody mix.  Note: this should take no more than 2 min
?. [Stain 1 (overnight)]
Add antibody master mix,
120 µl
The manufacturer recommend to use 100 µL per Sequenza slide assembly. 120 µL is therefore a 20% excess.
?. [Stain 1 (overnight)]
Place back the rack cover, transfer to 4˚C refrigerator, and incubate overnight
?. [Stain 2 (1h)]
Prepare 1 hr antibody master mix as described above in steps 15 to 18Optional: include use anti-biotin or other anti-hapten antibodies when apply
?. [Stain 2 (1h)]
Following overnight incubation, wash with 1 mL of wash buffer
?. [Stain 2 (1h)]
Repeat wash by 1 mL of wash buffer
?. [Stain 2 (1h)]
Add 300 µL antibody buffer (NHS 3%)
?. [Stain 2 (1h)]
Make sure that the antibody buffer has passed thru to prevent dilution of the antibody mix
?. [Stain 2 (1h)]
Add Stain 2 antibody mix and incubate for 1 hour at 4˚C
?. [Slide preparation]
The slide preparation is the same as described in the protocol MIBI staining dx.doi.org/10.17504/protocols.io.bh9zj976
?. [Wash and Post-fixation]
Add 1 mL of wash buffer
?. [Wash and Post-fixation]
Repeat step 29
?. [Wash and Post-fixation]
Remove the slide for the assembly
?. [Wash and Post-fixation]
Immerse the slide in post-fixation buffer (glutaraldehyde 4% in PBS-low Barium) for 5 min
?. [Wash and Post-fixation]
Transfer in PBS-low Barium for rinse,
?. [Dehydration]
Rearrange and fill additional reagent containers:Tris buffer pH 8.5 x 3, ddH2O x 2, 70% EtOH, 80% EtOH, 95% EtOH x 2, 100% EtOH x 2, exit tank = empty and dry
?. [Dehydration]
Insert slides into slide carriers. Place slide carriers into placeholder containers into the first Tris buffer container
?. [Dehydration]
Menu --> Processing time = 30 sec, Lift bar = 976, Number of dips = 3, Start position = position of the first slide carrier
?. [Dehydration]
Press Run
?. [Dehydration]
Dry in vacuum desiccator with the slide carrier lid open for at least 1 hour prior to MIBI analysis