TITLE: CODEX Antibody Staining Protocol for FFPE tissues
AUTHORS: Marda Jorgensen, Jerelyn Nick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the method for antibody staining of FFPE tissues on coverslips using CODEX Barcoded Antibodies.  Included are  the stepwise  protocols for pre-staining, deparaffinization, antigen retrival, antibody staining and post-fixation.  The enitre process requires approximately 6.5 hours, including a 3 hour incubation.   Except as noted, reagents and consumables should be prepared before being the protocol. Stained tissues can be stored in CODEX Storage Buffer at 4º C for up to 2 weeks for use in CODEX multiplex imaging.</div></div>

[STEPS]
?. [Tissue Pre-treatment         ent]
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.
55 °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 5.0 ml 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.
?. Place the sample coverslip(s) in the cover glass staining rack and wait 5 minutes to allow the tissue(s) to cool down.
?. [Deparaffinizationion]
Tissue Deparaffinization and Hydration Start the 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.
?. [Antigen Retrival rival]
In a 50 ml Pyrex Beaker, for each rack of slides, prepare 40 ml of 1x citrate buffer . Dilute 100x citrate buffer pH6.0 to 1X citrate buffer in ddH2O.
?. [Deparaffinizationion]
Immerse the staining rack in a staining container of Xylene covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a second staining container of Xylene covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a staining container of 100% Ethanol covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a second staining container of 100% Ethanol covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a staining container of 95% Ethanol covered for 5 minutes
?. [Deparaffinizationion]
mmerse the staining rack in a staining container of 90% Ethanol covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a staining container of 70% Ethanol covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a staining container of 50% Ethanol covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a staining container of 30% Ethanol covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a second staining container of ddH2O covered for 5 minutes.
?. [Deparaffinizationion]
Immerse the staining rack in a staining container of ddH2O covered for 5 minutes.
?. [Antigen Retrival rival]
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 rival]
Pour 1200 ml of DI water into the Instant Pot chamber and carefully place the sealed baker in the chamber.
?. [Antigen Retrival rival]
Close the Instant Pot lid.Set pressure to HIGH, timer for 20 minutes (turn off the keep warm setting). Press PRESSURE  COOK to start.
?. [Antigen Retrival rival]
After the cycle ends (about 35-40 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 until no longer hot to the touch.
Caution: beaker will be HOT when removed form pressure cooker!
?. [Antigen Retrival rival]
Place staining rack in a  40 ml beaker of ddH2O for a few seconds.
0 Room temperature
0 Room temperature
?. [Antigen Retrival rival]
Transfer staining rack to a second beakerr of 40 ml ddH2O, emerse for 10 minutes.
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 (STEP 14) carefully pick up each cover slip with bent tip forceps and place in prepared well #1 containing 5 ml of Hydration buffer. Incubate 5 seconds.
?. [Tissue Washing            ]
Move cover slip to Hydration buffer well #2. Incubate 5 seconds.
?. [Tissue Washing            ]
Move coverslip to the well containing 5 ml Staining Buffer. Incubate for 20-30 minutes. -
0 Room temperature
?. [Antibody Stainingng ]
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 3 hours. Do not disturb the chamber during incubation.
190 µl
0 Room temperature
?. [Post-Staining                ]
NOTE:  **At 2.5 hours of incubation:Add 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 (see STEP 4).  Incubate 2 minutes.
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 2-3 times.
?. [Post-Staining                ]
Transfer coverslip to PBS well #2. Immerge coverslip 2-3 times.
?. [Post-Staining                ]
Transfer cover slip to PBS well #3. Immerge coverslip 2-3 times.
?. [Post-Staining                ]
Transfer coverslip to well containing Ice cold methanol. Incubate for 5 minutes on ice.
on ice
?. [Post-Staining                ]
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 20 ul  of Final Fix to 1.0 ml of PBS. Mix thoroughly.]
?. [Post-Staining                ]
Reuse the filled PBS wells:  Quickly transfer coverslip to PBS well  #1. Avoid drying of the coverslip.
?. [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  a bottom labeled, well containing 5.0 ml Storage Buffer. Wrap edges of 6-well plate with parafilm and store at 4ºC. Record date on the cluster dish, stained tissue is stable for 2 weeks.
4 °C
Stained tissue can be stored  for up to 2 weeks.
?. 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 tray for holding pipette tips before placing it back in the box. Label different positions in the tray if working with multiple sample coverslips.Cover with the lid.
?. [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. Titer may require adjustment.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)