Feb 27, 2020
CODEX Antibody Staining Protocol for FFPE tissues
- 1University of Florida
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
Protocol Citation: Marda Jorgensen, Jerelyn Nick 2020. CODEX Antibody Staining Protocol for FFPE tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.bbsdina6
Manuscript citation:
Akoya BiosciencesInc,CODEX User Maual REV A.0 2019
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2020
Last Modified: February 27, 2020
Protocol Integer ID: 32293
Keywords: Akoya Biosciences, CODEX, antibody staining, FFPE, deparaffinization, Antigen Retrival, Post-fixation, CODEX Barcoded Antibodies,
Abstract
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.
Attachments
Guidelines
- The coverslips used to mount the tissue must be poly-l-Lysine coated.
- Take care to protect tissues from drying during transfer steps
- Always pipette at the corner of the coverslip and allow the liquid to flow over the tissue to minimize damage.
- Coverslips are fragile; use the reccommmended bent-tip forceps to handle the coverslips
- The hummidty chamber should be placed on a solid, vibration free table or bench top to maintain surface tension during the 3 hour incubation.
- 6-well plates can be washed and reused up to 5 times
Materials
CODEX® Staining Kit (Product Number: 7000008):
Stored at 4 C:
Hydration Buffer
Staining Buffer
Storage Buffer
N Blocker
G Blocker
Nuclear Stain
CODEX® Barcoded Antibodies
CODEX® Reporters
Stored at -20 C:
J Blocker
S Blocker
Fixative Reagent
Assay Reagent
Consumables & Glassware:
6-well plates Thermo14075
14 ml Tubes Thermo 339651
Screw cap vials/tubes 1.5 ml Fisherbrand02-681-372; 5.0 ml Fisherbrand 02-681-130
50 ml beaker (3) Fisherbrand 100-50
Equipment & Tools
Heating Plate that can be set to 55°C
Instant Pot IP-Duo 6 quart Model IPDuo60 V3
Eppendorf mini spin plus centrifuge for 1.5 ml tubes
Coverglass staining rack- 5 place ThermoFisher 116
Bent tip tweezers- Fine Science tools Dumont 1151-33 5/45C (recommended; no substitutions)
Humidity Chamber (homemade) empty ART 1.0 ml pipette tip box with lid
Solvents and Buffers
Xylene Fisher X3P-1GAL
Ethanol 200 proof Decon Lab 2701
Methanol Acros 61009-0040
16% formaldehyde Solution (methanol free) Thermo 28906
DPBS 1X Gibco 14190-44
Nuclease-Free water (not DEPC treated) Ambicon AM9938
Citrate Buffer pH 6.0 100X Abcam ab64236
Safety warnings
Use of a fume hood is recommended for working with flammable solvents. Methanol should be kept in a refrigerator approved for storage of flammable liquids. Xylene, ethanol, PFA and methanol must be disposed of in the appropriate hazardous waste containers.
Before start
Prepare reagents, except as noted, and consumables before beginning protocol.
Tissue Pre-treatment ent
Tissue Pre-treatment ent
30m
30m
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
00:20:00 -00:25:00
Note
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.
6-well plates for 2 tissues
Note
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.
Hummidity Chamber
Place the sample coverslip(s) in the cover glass staining rack and wait 5 minutes to allow the tissue(s) to cool down.
00:05:00
Deparaffinizationion
Deparaffinizationion
1h
1h
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. 100 mL each 00:05:00 each step
Safety information
Note: It is highly recommended that you perform this procedure in a fume hood; organic solvents are toxic and highly volatile.
Immerse the staining rack in a staining container of Xylene covered for 5 minutes.
5m
Immerse the staining rack in a second staining container of Xylene covered for 5 minutes.
Immerse the staining rack in a staining container of 100% Ethanol covered for 5 minutes.
Immerse the staining rack in a second staining container of 100% Ethanol covered for 5 minutes.
Immerse the staining rack in a staining container of 95% Ethanol covered for 5 minutes
mmerse the staining rack in a staining container of 90% Ethanol covered for 5 minutes.
Immerse the staining rack in a staining container of 70% Ethanol covered for 5 minutes.
Immerse the staining rack in a staining container of 50% Ethanol covered for 5 minutes.
Immerse the staining rack in a staining container of 30% Ethanol covered for 5 minutes.
Immerse the staining rack in a staining container of ddH2O covered for 5 minutes.
Immerse the staining rack in a second staining container of ddH2O covered for 5 minutes.
Antigen Retrival rival
Antigen Retrival rival
40m
40m
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.
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.
Pour 1200 ml of DI water into the Instant Pot chamber and carefully place the sealed baker in the chamber.
Close the Instant Pot lid.Set pressure to HIGH, timer for 20 minutes (turn off the keep warm setting). Press PRESSURE COOK to start.
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.
00:40:00
Safety information
Caution: beaker will be HOT when removed form pressure cooker!
Place staining rack in a 40 ml beaker of ddH2O for a few seconds.
Room temperature Room temperature
Transfer staining rack to a second beakerr of 40 ml ddH2O, emerse for 10 minutes.
00:10:00 Room temperature
Note
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.]
Room temperature
| CODEX BB | 2 samples | 3 samples | 4 samples | 6 samples | 10 samples | |
| Staining buffer | 362 ul | 543 | 724 | 1086 | 1810 | |
| N Blocker | 9.5 ul | 14.25 | 19 | 28.5 | 47.5 | |
| G Blocker | 9.5 ul | 14.25 | 19 | 28.5 | 47.5 | |
| J Blocker | 9.5 ul | 14.25 | 19 | 28.5 | 47.5 | |
| S Blocker | 9.5 ul | 14.25 | 19 | 28.5 | 47.5 | |
| Total | 400 ul | 600 | 800 | 1200 | 2000 |
CODEX BLOCKING BUFFER by number of tissue samples
Tissue Washing
Tissue Washing
30m
30m
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.
00:00:05
Move cover slip to Hydration buffer well #2. Incubate 5 seconds.
00:00:05
Move coverslip to the well containing 5 ml Staining Buffer. Incubate for 20-30 minutes.
00:20:00 -00:30:00
Room temperature
Note
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 volume
Final 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 Stainingng
Antibody Stainingng
3h
3h
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 03:00:00 Room temperature
Post-Staining
Post-Staining
1h 30m
1h 30m
Note
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).
On ice
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 Room temperature
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.
00:02:00 Room temperature
Transfer coverslip to staining buffer well #2 of the second cluster dish. Incubation 2 minutes.
00:02:00 Room temperature
Transfercoverslip to PFA well. Incubate 10 minutes.
00:10:00 Room temperature
Transfer coverslip to PBS well #1. Immerge coverslip 2-3 times.
Transfer coverslip to PBS well #2. Immerge coverslip 2-3 times.
Transfer cover slip to PBS well #3. Immerge coverslip 2-3 times.
Transfer coverslip to well containing Ice cold methanol. Incubate for 5 minutes on ice.
On ice
00:05:00
Note
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.]
Room temperature
Reuse the filled PBS wells: Quickly transfer coverslip to PBS well #1. Avoid drying of the coverslip.
Immediately transfer coverslip to PBS well #2.
Immediately transfer coverslip to PBS well #3
Final Fix
Final Fix
20m
20m
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 00:20:00 Room temperature
Reuse PBS wells: Transfer coverslip to PBS well #1.
Immediately transfer coverslip to PBS well #2.
Immediately transfer cover slip to PBS well #3
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
Note
Stained tissue can be stored for up to 2 weeks.