TITLE: HuBMAP TMC-UF Validation of Custom conjugated Antibodies for CODEX
AUTHORS: Marda Jorgensen, Jerelyn Nick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the methods for validating antibodies after custom conjugation with Akoya CODEX barcodes in FFPE tissues on coverslips. Included are the stepwise  protocols for pre-staining, deparaffinization, antigen retrival, antibody staining, post-fixation, manual addition of CODEX Reporters and tissue mounting on microscope slides.  The recommended single stain experimental design includes 3 tissues sections from the same block stained with the CODEX-tagged antibody alone (1), co-stained with a positive control(2), and co-satined with a negative control(3).  </div><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td><td>C</td><td>D</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;"> </td><td rowspan = "" colspan = "" style ="display : table-cell;">Tissue 1</td><td rowspan = "" colspan = "" style ="display : table-cell;">Tissue 2</td><td rowspan = "" colspan = "" style ="display : table-cell;">Tissue 3</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;"> </td><td rowspan = "" colspan = "" style ="display : table-cell;">CODEX-Stain Only&nbsp;</td><td rowspan = "" colspan = "" style ="display : table-cell;">CODEX-stain
  with co-stain (Positive Control)&nbsp;</td><td rowspan = "" colspan = "" style ="display : table-cell;">CODEX-stain
  with counter-stain (Negative Control)</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">DAPI Channel</td><td rowspan = "" colspan = "" style ="display : table-cell;">Nuclear Stain</td><td rowspan = "" colspan = "" style ="display : table-cell;">Nuclear Stain</td><td rowspan = "" colspan = "" style ="display : table-cell;">Nuclear Stain</td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">2nd
  Fluorescent Channel</td><td rowspan = "" colspan = "" style ="display : table-cell;">CODEX-tagged Anitbody</td><td rowspan = "" colspan = "" style ="display : table-cell;">CODEX-tagged Anitbody</td><td rowspan = "" colspan = "" style ="display : table-cell;">CODEX-tagged Anitbody</td></tr><tr><td style = "text-align:center;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">3rd or 4th
  Fluorescent Channel</td><td rowspan = "" colspan = "" style ="display : table-cell;">None&nbsp;</td><td rowspan = "" colspan = "" style ="display : table-cell;">Control
  antibody targeting a different antigen expressed by the same phenotype or
  cell population</td><td rowspan = "" colspan = "" style ="display : table-cell;">Control
  antibody targeting an antigen expressed by a different phenotype or cell
  population.</td></tr></table><div class = "text-block">The enitre process requires approximately 5.5 hours, including a 2 hour incubation.   Except as noted, reagents and consumables should be prepared before begining the protocol. </div></div>

[STEPS]
?. [Tissue Pre-treatment     ]
Turn on the heating plate and set it at 55°C.
55 °C
?. Once the heating plate has reached 55°C, retrieve the FFPE samples on  poly-l-Lysine treated coverslip(s) from 4°C storage.
4 °C
?. Using bent tip forceps, place the sample coverslip(s) on the hot plate with the tissue facing up. Incubate 20-25 minutes until wax thoroughly melts. -
55 °C
?. NOTE:**[While wax is melting from tissue on coverslips, label and fill 6-well plates.  Add    of reagent per well. Each coverslip requires two wells of Hydration Buffer (H),  three of Staining Buffer (S)  and three of DPBS. Allow reagents to warm to room temperature. One well each of PFA and ice cold methanol will be filled later in the protocol. Store the empty methanol plate at 4C.
?. NOTE: **[While wax is melting from tissue on coverslips, create Humidity Chamber from an empty  pipette tip box with lid. Wet a paper towel and place it at the bottom of the pipette box. Fill the pipette box with enough ddH2O at the bottom to fully cover the paper towel (ca. 1-2 cm deep). Rinse and dry the top tray before placing it back in the box. Label different positions in the tray if working with multiple sample coverslips.Cover with the lid.
?. After 20 minute incubation on warmer, place the sample coverslip(s) in the cover glass staining rack and wait 5 minutes to allow the tissue(s) to cool down.
?. [Deparaffinization]
Tissue Deparaffinization and Hydration Start the deparafffinization and rehydration process by placing the coverglass staining rack in the following solvent series. Each incubation step lasts for 5 minutes. Make sure the coverslip(s) are completely covered by the liquid and move the rack gently at start of each new step to make sure the liquid in the space between coverslips is exchanged. Close the containers with lids during incubation.
[each]
[each step]
Note: It is highly recommended that you perform this procedure in a fume hood; organic solvents are toxic and highly volatile.
?. [Deparaffinization]
Immerse the staining rack in a staining container of Xylene covered for
?. [Deparaffinization]
Immerse the staining rack in a second staining container of Xylene covered for
?. [Deparaffinization]
Immerse the staining rack in a staining container of 100% Ethanol covered for
?. [Deparaffinization]
Immerse the staining rack in a second staining container of 100% Ethanol covered for
?. [Deparaffinization]
mmerse the staining rack in a staining container of 90% Ethanol covered for
?. [Deparaffinization]
Immerse the staining rack in a staining container of 70% Ethanol covered for
?. [Deparaffinization]
Immerse the staining rack in a staining container of 50% Ethanol covered for
?. [Deparaffinization]
Immerse the staining rack in a staining container of 30% Ethanol covered for
?. [Deparaffinization]
Immerse the staining rack in a staining container of ddH2O covered for
?. [Deparaffinization]
Immerse the staining rack in a second staining container of ddH2O covered for
?. [Antigen Retrival ]
In a 50 ml pyrex beaker, for each rack of slides, prepare   of 1x citrate buffer . Dilute 100x citrate buffer pH6.0 to 1X citrate buffer in ddH2O.
40 mL
?. [Antigen Retrival ]
Immerse the staining rack(s) in the beaker(s) containing the 1x citrate buffer and wrap tightly with aluminum foil to ensure the best sealing possible. Seal well around the rim and spout of the beaker to prevent water vapor from entering the beaker.
?. [Antigen Retrival ]
Pouro f DI water into the Instant Pot chamber and carefully place the sealed beaker in the chamber.
1200 mL
?. [Antigen Retrival ]
Close the Instant Pot lid.Set pressure to HIGH, timer for   (turn off the keep warm setting). Press PRESSURE  COOK to start.
?. [Antigen Retrival ]
After the cycle ends (about 40-45 min), slowly release the pressure cooker vent. Using a hot mit, and lifting from beaker rim, carefully take the rack out from the pressure cooker. Allow to cool on the bench briefly.Step 12  about .   total.
Caution: beaker will be HOT when removed form pressure cooker!
?. [Antigen Retrival ]
Place staining rack in a  50 ml beaker containing    of ddH2O for a few seconds to cool.
40 mL
0 Room temperature
0 Room temperature
?. [Antigen Retrival ]
Transfer staining rack to a second beakerr containing   ddH2O, emerse for
40 mL
0 Room temperature
?. ABCDEF1  CODEX BB    2 samples    3 samples    4 samples    6 samples     10 samples  2  Staining buffer    362 ul    543    724       1086    1810  3  N Blocker    9.5 ul    14.25    19    28.5    47.5  4  G Blocker    9.5 ul    14.25    19    28.5    47.5  5  J Blocker    9.5 ul    14.25    19    28.5    47.5  6  S Blocker    9.5 ul    14.25    19    28.5    47.5  7  Total    400 ul    600    800    1200    2000
NOTE: **[During STEP 14, make CODEX Blocking Buffer by adding Blockers N, G, J, and S to staining buffer according to the number of samples/coverslips you are staining.]
ABCDEF1  CODEX BB    2 samples    3 samples    4 samples    6 samples     10 samples  2  Staining buffer    362 ul    543    724       1086    1810  3  N Blocker    9.5 ul    14.25    19    28.5    47.5  4  G Blocker    9.5 ul    14.25    19    28.5    47.5  5  J Blocker    9.5 ul    14.25    19    28.5    47.5  6  S Blocker    9.5 ul    14.25    19    28.5    47.5  7  Total    400 ul    600    800    1200    2000
?. [Tissue Washing            ]
After 10 min incubation in water (STEP 14) carefully pick up each cover slip with bent tip forceps and place in prepared well #1 containing   of Hydration buffer. Incubate 5 seconds.
5 mL
?. [Tissue Washing            ]
Move cover slip to Hydration buffer well #2. Incubate 5 seconds.
?. [Tissue Washing            ]
Move coverslip to the well containing  l Staining Buffer. Incubate for 20-30 minutes. -
5 mL
0 Room temperature
Do not exceed   in Staining Buffer
?. [Tissue Washing            ]
NOTE: **[During STEP 18, prepare Antibody Cocktail by adding  the primary barcoded antibodies to the Codex Blocking Buffer prepared in STEP 15. ] Akoya inventoried antibodies are applied at 1ul per tissue stained (1:200 dilution) . Titer may require adjustment.  Newly conjugate antibodies are typically tested at 1, 2 or 4 ul per tissue (dilution of 1:200, 1:100, 1:50, respectively)If standard fluorescent antibodies are being used as co- and counter-stains thay should be added here in the predetermined dilution.Subtract the total volume of antibodies from the volume of CODEX blocking buffer prepared __­­________ #Primaries @ 1ul per tissue X __­­­­____# Tissues= _________ul total Primary volumeFinal antibody cocktail will be:Antibody Cocktail___________ ul volume Blocking Buffer (STEP 15)-__________ ul total primary volume (calculted above)=__________ul adjusted volume of Blocking Buffer+__________ul total Primary @ 1ul per tissue=__________ul Final volume of Primary cocktail ( will be equivalent to initial volume of Blocking Buffer)
?. [Antibody Staining ]
At the completion of STEP 18 add antibody cocktail to the coverslips.Working one tissue at a time, remove coverslip from staining buffer with bent tip forceps, tip to drain, and place on humidity chamber. Immediately add 190 ul of antibody cocktail to a corner of the coverslip. Avoid pipetting directly over tissue. Avoid bubbles. Repeat for each coverslip. Cover the humidity chamber with lid and incubate at room temperature for 2 hours. Do not disturb the chamber during incubation.
190 µl
0 Room temperature
?. [Post-Staining                ]
NOTE:  **At 1.5 hours of incubation: Prepare Methanol and PFAAdd 5.0 ml ice cold  methanol to the prelabled  6-well plate, one well is needed  for each coverslip. Set plate on ice (See STEP 4). Prepare the PFA solution. To 9 mls of storage buffer add 1.0 ml of 16% PFA. For each coverslip, place 5.0 ml into the well of a labeled  6-wellplate.  see STEP 4
?. [Post-Staining                ]
After antibody incubation is completed: Using bent tip forceps, carefully lift the coverslip out of the humidity chamber, tip to drain antibody solution and place in   Staining Buffer well #1 of the second cluster dish containing (see STEP 4).  Incubate 2 minutes.
5 mL
0 Room temperature
?. [Post-Staining                ]
Transfer coverslip to Staining Buffer well #2 of the second cluster dish. Incubation 2 minutes.
0 Room temperature
?. [Post-Staining                ]
Transfercoverslip to PFA well. Incubate 10 minutes.
0 Room temperature
?. [Post-Staining                ]
Transfer coverslip to PBS well #1. Immerge coverslip
?. [Post-Staining                ]
Transfer coverslip to PBS well #2. Immerge coverslip
?. [Post-Staining                ]
Transfer cover slip to PBS well #3. Immerge coverslip .
?. [Post-Staining                ]
Transfer coverslip to well containing Ice cold methanol. Incubate for 5 minutes on ice.
on ice
?. [Post-Staining                ]
Prepare Final Fix Solution
NOTE:  **[Wash/dry the staining chamber and prepare Final Fix solution, near the end of  STEP 28. Rapidly thaw CODEX Final Fix Reagent and spin down briefly. Add   of Final Fix to   of PBS.  Mix thoroughly.
?. [Post-Staining                ]
Reuse the filled PBS wells:  Quickly transfer coverslip to PBS well  #1. Avoid drying of the coverslip.
Tissues in methanol tend to dry out quickly
?. [Post-Staining                ]
Immediately transfer coverslip to PBS well #2.
?. [Post-Staining                ]
Immediately transfer  coverslip to PBS well #3
?. [Final Fix               ]
Using bent tip forceps, carefully transfer coverslips one at a time to the cleaned humidity chamber. Immediately add 190 ul of the final fix solution to the corner of the coverslip. Repeat with each coverslip. Incubate for 20 minutes.
190 µl
0 Room temperature
?. [Final Fix               ]
Reuse PBS wells: Transfer coverslip to PBS well #1.
?. [Final Fix               ]
Immediately transfer coverslip to PBS well #2.
?. [Final Fix               ]
Immediately transfer cover slip to PBS well #3
?. [Final Fix               ]
Place each coverslip tissue side up in  labeled well of 6-well plate containing   ml Storage Buffer. Wrap edges of 6-well plate with parafilm and store at 4ºC.Or proceed to manual addition of CODEX reporter.
5 mL
4 °C
Stained tissue can be stored  for up to 2 weeks.
?. [Manual Addition of CODEX Reporter]
Prepare Screening Buffer in a glass beaker. For 3 coverslips use:Screening Buffer  3.5 ml 10X CODEX Buffer24.5 ml ddH2O  7.0 ml DMSO
35 mL
Screening Buffer should be prepared fresh for each experiment.  This process is exothermic. Allow to equillibatrate for 20 minute prior to use
?. [Manual Addition of CODEX Reporter]
Add  Screening Buffer to 6-well plate.  3 wells per tissue. Also, add   1X CODEX Buffer to 6-well plate, 1 well per coverslip.
5 mL
5 mL
0 Room temperature
?. [Manual Addition of CODEX Reporter]
Place coverslip in well #1 Screening Buffer
?. [Manual Addition of CODEX Reporter]
Move cover slip to well #2 Screening Buffer
?. [Manual Addition of CODEX Reporter]
Move cover slip to Well #3 Screening Buffer
Coversplips can be left in Screening Buffer for MAX of
?. [Manual Addition of CODEX Reporter]
Prepare 1X CODEX Buffer. For 3 coverslips prepare at least Nuclease Free Water   10x CODEX Buffer
20 mL
18 mL
2 mL
?. [Manual Addition of CODEX Reporter]
Prepare Reporter Stock Solution for 3 sample coverslips1X CODEX Buffer  Assay Reagent            Nuclear Stain
284.5 µl
15 µl
1 µl
?. [Manual Addition of CODEX Reporter]
Prepare Reporter Master Mix. Thaw reporters, if frozen, and spin briefly. Label 3 amber eppendorf tube for each experiment.  Add Reporter Stock Solution according to the number of  CODEX tagged antibodies/Reporters per sample coverslip.  add 2.5 ul of each Reporter.  Reporter Master Mix final violume  .1  coverslip   +  1 reporter  =   Reporter Stock solution  +   Reporter1 coverslip    +  2 reporters=       Reporter Stock solution  +   EACH of 2 Reporters
100 µl
97.5 µl
2.5 µl
95 µl
2.5 µl
[ Reporter Master Mix]
Use amber colored eppendorf tubes to protect fluorescent reporter stock solutions from light.
?. [Incubate Sample coverslip with CODEX Reporters]
Tape a square of parafilm to the benchtop.  Carefully pipette   of each Reporter master Mix onto the paraflim
95 µl
?. [Incubate Sample coverslip with CODEX Reporters]
Remove the sample cover slip from the screening bufferINVERT the coverslip onto the corresponding droplet of Reporter master Mix
Place coverslip down by gently placing one edge first and then slowly lowering the coverslip on top of the liquid.
?. [Incubate Sample coverslip with CODEX Reporters]
Cover samples with aluminum foil or a small box to prevent light exposure. Incubate . Change gloves.
0 Room temperature
Protect reporters from light to prevent photobleaching
?. [Incubate Sample coverslip with CODEX Reporters]
Invert coverslip so that tisssue is facing up.  Place in well #1 Screening Buffer.
?. [Incubate Sample coverslip with CODEX Reporters]
Move coverslips to well #2 Screening Buffer
?. [Incubate Sample coverslip with CODEX Reporters]
Mover coverslip to well # 3 Screening Buffer
?. [Incubate Sample coverslip with CODEX Reporters]
Move coverslip to Well containing 1X CODEX Buffer
?. [Mount Tissue ]
Label 3 microscope slides, one for each coverslip.
?. [Mount Tissue ]
Cut the end off of a 200 ul pipette tip.  Add   of Fluoromount-G  to each slide.  Remove coverslip from 1X CODEX buffer and INVERT onto mountant.
15 µl
Take  care not to trap bubbles under coverslip. If bubbles form, remove tissue coverslip immerse in 1X CODEX buffer and repeat mounting procedure.
?. [Mount Tissue ]
Blot coverslip edges on kimwipe to remove excess liquid.  cover edges with nail polish to seal.  Allow 5-7 minutes to dry in the dark.
?. [Mount Tissue ]
After sealant has dried, remove buffer salts from top of the sample coverslip with a wet Kimwipe.
?. [Mount Tissue ]
Image with  the fluorescent microscope associated with the CODEX Instrument using the same settings used for CODEX.  For example, all 4 channels, 20X magnification with the same acquisition times.