Apr 01, 2022
CODEX Sample Preparation and Staining Experiment Protocol
- 1UCSF
Protocol Citation: Kavya.Anjani, Miguel Rivera, Zoltanlaszik 2022. CODEX Sample Preparation and Staining Experiment Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk4pmxg5r/v1
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: August 05, 2021
Last Modified: April 03, 2022
Protocol Integer ID: 52125
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
CODEX system is the combination of an (1) oligo-nucleotide based antibody labeling-detection technique, (2) a microfluidics instrument coupled with an inverted microscope capable of whole slide scanning, and an (3) ImageJ-based analysis platform. This experimental setup allows the automation of immunofluorescence signal detection across several staining cycles of a single tissue section
This protocol describes detailed methods used in CODEX sample preparation and staining at the Laszik Lab.
Guidelines
QA/QC Parameters
1. Visual inspection of whole slide digital images of the stains. Every stain (channel-target) in each section is visually evaluated.
2. Positive controls. A positive control stain is performed with each CODEX stain on a “control” kidney with inflammation.
3. Automation. Using standardized operator-independent automated validated protocols.
CODEX data quality management processing
All CODEX data generated go through a rigorous quality control process that includes visual assessment of the processed images and a review of the segmented files, both test specimens and on slide controls. In addition to the stains, cellular and compartmental segmentation fidelity is key to downstream analysis. Unexpected staining pattern and/or artifacts on the processed images are documented; if correctable, such as those related to folding or edge artifact among others, the images are manually annotated to define regions that could be rescued for analytics. If corrective measures are not an option, such as that with a failed stain the data for that particular stain will be excluded from the analysis. Segmented images, both with cellular and compartmental segmentation are also reviewed for accuracy; once again, if abnormal/unexpected segmentation is detected, depending on the nature of such finding either corrective measures are implemented or the case/stain is flagged and excluded from further analysis.
Materials
CODEX staining
Antibodies, barcodes, reporters
- CODEX barcoded antibodies
- CODEX barcodes
- CODEX reporters
CODEX Staining Kit
- Staining Buffer
- Storage Buffer
- N Blocker
- G Blocker
- J Blocker
- S Blocker
- Fixative reagent
Consumables, solvents, equipment, glassware, biologics, reagents, instrumentation
- 10X CODEX® Buffer
- CODEX® Gaskets
- Coverslips
- Tissue Processing
- CODEX® Run 96 well plates
- 96 well plate seals
- Assay Reagent
- Nuclear Stain
- Glass beaker (0.5 L) 6 X Glass beakers (50 mL)
- Buffer reservoir
- Buffer tray - Required. No Substitutions. VWR
- Saran wrap
- Plastic petri dish
- Coverslip Prep Cardboard freezer box
- Lab tape
- Bent-tip tweezers
- 6-Well TC Plates - Does not need to be tissue cultured treated.
- 1mL, 1.5 mL, 2 mL
- Amber 1.5 mL tubes
- 5, 15, 50 ml conical tubes
- 16% Paraformaldehyde Staining Tissue
- 1X PBS
- Conjugation,
- Poly-L-Lysine 0.1%
- Drierite Absorbents
- Nuclease-Free Water
- Fluoromount-G™ (optional)
- MilliQ H2O
- Acetone
- Methanol
- DMSO - ACS reagent, ≥99.9%
- CODEX instrument
- CODEX software suite
CODEX Conjugation
CODEX Conjugation Kit
- Filter Blocking Solution
- Reduction Solution 1
- Reduction Solution 2
- Conjugation Solution
- Purification Solution
- Antibody Storage Solution
Consumables, solvents, equipment, glassware, biologics, reagents, instrumentation
- 50kDa MWCO filter
- Screw-top 1 or 2mL tubes
- Customer choice
- Parafilm
- NuPAGE™ LDS Sample Buffer (14X)
- NuPAGE™ Sample Reducing Agent (10X)
- Conjugation QC
- NuPAGE™ 4-12% Bis-Tris Protein Gels Thermo Fisher Scientific NP0321BOX Conjugation QC
- Novex™ Sharp Pre-Stained Protein Standard 3.5-260 kDa
- Novex™ SimplyBlueTM SafeStain NuPAGE™ MOPS SDS Running Buffer (20X)
- XCell SureLock™ Mini-Cell Electrophoresis System
- 95°C dry bath
- Nanodrop
- Shaker
- Microwave
Tissue Sample Preparation Prior to CODEX Staining
Tissue Sample Preparation Prior to CODEX Staining
12h 1m
12h 1m
Gather necessary materials
Note
Materials needed:
- Coverslips (CODEX Mounting Kit 1.0 @RT)
- Poly-Lysine Solution 1% (CODEX Mounting Kit 1.0 @RT)
- 600 mL glass beaker
- Rubber band
- Saran wrap
- Milli-Q water
- Plastic petri dish
- Paper towels
Coat coverslips with poly-lysine.
Note
*Incubating the coverslips in poly-lysine will need to take place least 12 hours prior to embedding tissue samples*
Gently place 20 coverslips on the bottom of the glass beaker and slowly swirl the beaker to fan out the coverslips.
Add 70 mL of poly-lysine solution to the beaker, making sure to submerge all coverslips.
To prevent evaporation, use the saran wrap to cover the beaker and seal the wrap with a rubber band.
STOPPING POINT- Incubate coverslips in poly-lysine solution for a minimum of 12:00:00 and up to one week at Room temperature .
12h
After incubation, dispose of the poly-lysine solution into a proper waste container.
Fill the beaker halfway with Milli-Q water and swirl contents to rinse coverslips.
Let the beaker and coverslips sit for 00:01:00 .
1m
Dispose of the water into the sink.
Repeat steps 2.5-2.8 for a total of 7 washes.
Fill the beaker halfway with Milli-Q water.
Place two sets of paper towels on the benchtop.
Remove the coverslips from the beaker, then separate and lay each piece face down on the first set of paper towels.
Invert each coverslip to dry the reverse side on the second set of paper towels.
Once the coverslips dry completely, collect them in a plastic petri dish to store for up to 2 months.
Cut and embed frozen tissues on poly-lysine coated coverslips.
Place poly-lysine coated coverslips in the cryostat chamber to equilibrate.
Cut frozen tissue slices with 5 µm - 10 µm thickness.
Note
Do not exceed 10 µm
Gently place tissue slice in the center of the coverslip.
Place a gloved finger on the underside of the coverslip behind the tissue slice to melt the OCT for better adherence. Do not exceed 2 seconds.
Store tissue slices separated at-80 °C for up to 6 months prior to staining.
Antibody Conjugation with CODEX-tags for Custom Panel
Antibody Conjugation with CODEX-tags for Custom Panel
5h 14m 5s
5h 14m 5s
Gather necessary materials.
Note
Material needed:
- Antibody Reduction Solution 2 (CODEX Conjugation Kit 1.0 @ 4C)
- Antibody Conjugation Solution (CODEX Conjugation Kit 1.0 @ 4C)
- Filter Blocking Solution (CODEX Conjugation Kit 1.0 @ 4C)
- Antibody Reduction Solution 1 (CODEX Conjugation Kit 1.0 @-20C, thaw right before use, one tube for every 3 antibodies)
- CODEX Antibody Tags (CODEX Conjugation Kit 1.0 @-20C, thaw right before use)
- Antibody Purification Solution (CODEX Conjugation Kit 1.0 @ 4C)
- Antibody Storage Solution (CODEX Conjugation Kit 1.0 @ 4C)
- Purified Antibodies (50 μg needed per antibody)
- 50 kDa MWCO filter
- 2 mL screw-top tubes
- Milli-Q water
Calculate and obtain 50 µg of each antibody that will be conjugated.
Label a 50kDA MWCO filter unit (both the filter device and the collection tube) for each antibody that will be conjugated.
Add 500 µL of Filter Block Solution to the filter device.
Spin down the filter unit at 12000 x g, 00:02:00 .
2m
Discard all liquid (from both the filter device and the collection tube).
Add 50 µg of antibody to the filter device.
Spin down the filter unit at 12000 x g, 00:08:00 .
8m
Prepare the Antibody Reduction Master Mix while spinning.
Note
For one antibody, mix the following:
6.6 µL Antibody Reduction Solution 1 + 275 µL Antibody Reduction Solution 2
Multiply the solution 1 and solution 2 volumes by the total number of antibodies that will be conjugated to get the total volume needed from each reagent.
Discard flow through.
Add 260 µL of Antibody Reduction Master Mix to each filter device.
Vortex solution in filter device for00:00:02 - 00:00:03 .
5s
Incubate tube for 00:30:00 at Room temperature .
30m
Spin down the filter unit at 12000 x g, 00:08:00 .
8m
Discard flow-through.
Add 450 µL of Antibody Conjugation Solution to each filter device.
Spin down the filter unit at 12000 x g, 00:08:00 .
8m
Prepare the CODEX antibody tag solution while spinning.
Label a new tube for each CODEX antibody tag (one tag per antibody).
Add 210 µL of Antibody Conjugation Solution to each tube in step a. Set aside.
Add 10 µL of Milli-Q water to resuspend each CODEX antibody tag (retrieved from -20 just prior). Mix by pipetting.
Add the entire resuspended CODEX antibody tag to step b and mix by pipetting.
Discard flow-through.
Add the CODEX antibody tag solution prepared in step 21 to the corresponding filter device.
Close the lid and vortex the filter unit for 2-3 sec.
Incubate tube for 02:00:00 at Room temperature .
2h
After 02:00:00 , set aside 5 µL of the purified solution for QC.
2h
Spin down the filter unit at 12000 x g, 00:08:00 .
8m
Discard flow-through.
Add 450 µL of Antibody Purification Solution to each filter device.
Spin down the filter unit at 12000 x g, 00:08:00 .
8m
Discard flow-through.
Repeat steps 29-31 two more times for a total of three times.
There will be solution left in the filter device.
Label a new collection tube for each filter device.
Add 100 µL Antibody Storage Solution to each filter device.
Invert the filter device into its newly labeled collection tube.
Spin down the filter unit at3000 x g, 00:02:00 .
2m
Transfer solution in the collection tube to a labeled 2 mL screw-top tube and store at 4 °C for up to 1 year.
Tissue Staining
Tissue Staining
5h 14m 5s
5h 14m 5s
Gather necessary materials.
Note
Materials needed:
- Hydration Buffer (CODEX Staining Kit 1.0 @ 4C)
- Staining Buffer (CODEX Staining Kit 1.0 @ 4C)
- Storage Buffer (CODEX Staining Kit 1.0 @ 4C)
- Blocking Component N (CODEX Staining Kit 1.0 @ 4C)
- Blocking Component J (CODEX Staining Kit 1.0 @ 4C)
- Blocking Component G (CODEX Staining Kit 1.0 @ -20C)
- Blocking Component S (CODEX Staining Kit 1.0 @ -20C)
- CODEX-Tagged Antibodies (CODEX Antibody Kit 1.0 @ -20C)
- Fixative (F) Aliquots (CODEX Staining Kit 1.0 @ -20C, thaw right before use, one tube for every 5 tissue samples)
- Acetone
- Methanol (Keep in -20C prior to starting experiment)
- 16% PFA
- 1 x DPBS
- 6-well TC plates
- Bent-tip tweezers
- Drierite Absorbents
- Eppendorf tubes
- Conical tubes
- 50 mL beakers
- Empty pipet tip boxes
Prepare a humidity chamber.
Place wet paper towels at the bottom of an empty pipet tip box.
Fill the box with enough water to cover the towels completely.
Recover the pipet tip tray and lid after rinsing.
Prepare Drierite absorbent beads.
Fill an empty pipet tip box with Drierite beads (about 1~2 cm deep).
Prepare the following plate configuration for CODEX solutions:
*5 mL of each solution per well
Prepare 10 mL of acetone in a 50 mL beaker for each tissue.
Remove the tissue sections from -80C and place the coverslips on Drierite absorbent beads.
Let the tissue sections dry for 00:02:00 at Room temperature .
2m
Place the tissue samples face-up in the beakers containing acetone.
Incubate for 00:10:00 .
10m
Remove the coverslips from acetone and place them in the humidity chamber to dry for 00:02:00 at Room temperature .
2m
Hydrate tissue samples following the plate configuration.
Place the coverslips in the Hydration Buffer wells (dip 2-3 times before fully submerging the slide). Ensure tissue is facing up.
Incubate for 00:02:00 .
2m
Place the coverslips in the next set of Hydration Buffer wells.
Incubate for00:02:00 .
2m
Prepare Pre-Staining Fixing Solution while incubating tissue.
Note
For two samples, mix the following:
1 mL 16% PFA + 9 mL Hydration Buffer
Multiply the PFA and buffer volumes by the total number of samples that will be stained to get the total volume needed from each reagent. Add5 mL of Fixing Solution 1 to each well.
Place the coverslips in the Pre-Staining Fixing Solution wells.
Incubate for 00:10:00 at Room temperature .
10m
Remove samples from Pre-Staining Fixing Solution and rinse the samples by dunking the coverslips 2-3 times each in the top and bottom Hydration Buffer wells.
Place the samples in the Staining Buffer wells and let equilibrate for 00:20:00 - 00:30:00 .
50m
Prepare the antibody cocktail during equilibration.
To make CODEX Blocking Buffer for two samples, mix the following:
10 µL Blocking Component N + 10 µL Blocking Component G + 10 µL Blocking Component J + 10 µL Blocking Component S + 380 µL Staining Buffer
Multiply the reagent volumes by the total number of samples that will be stained to get the total volume needed for the experiment.
Calculate the total antibody volume per antibody cocktail.
Total Ab volume = (# of antibodies) x (# of samples) x (Ab volume/sample)]
Note
E.g. To stain two tissue samples with 24 antibodies and assuming 1 μL of antibody is needed per sample:
Total Ab volume = 24 antibodies x 2 samples x 1 μL/sample = 48 μL
Calculate to CODEX Blocking Buffer needed per antibody cocktail.
CODEX Blocking Buffer volume needed to make 210 μL Ab Cocktail Solution/sample = CODEX Blocking Buffer volume - Total Ab volume
Note
E.g. For two tissue samples with a total Ab volume of 48 μL:
CODEX Blocking Buffer volume needed for Ab Cocktail Solution = 420 μL Blocking Buffer volume - 48 μL total Ab volume = 372 μL
-> Ab Cocktail Solution = 48 μL Ab volume + 372 μL CODEX Blocking Buffer
Combine the antibodies and the CODEX Blocking Buffer to make 210 µL Antibody Cocktail Solution/tissue sample.
Remove the coverslips from Staining Buffer and place in the humidity chamber.
Quickly add 200 µL of Antibody Cocktail Solution to each sample from the top corner of the coverslip.
Place lid back on the humidity chamber.
Incubate for 03:00:00 at Room temperature .
3h
Prepare the following plate configuration for CODEX solutions ~1 hour before the 3-hour incubation ends:
*5 mL of each solution per well. Schematic is for two tissue samples.
Wash the tissue samples following the plate configuration.
Place the coverslips in the Staining Buffer (SB) wells (dip 2-3 times before fully submerging the slide). Ensure tissue is facing up.
Incubate for 00:02:00 .
2m
Place the coverslips in the next set of Staining Buffer (SB) wells.
Incubate for 00:02:00 .
2m
Prepare Post-Staining Fixing Solution (PS) while incubating tissue.
For two samples, mix the following:
1 mL 16% PFA + 9 mL Storage Buffer
Multiply the PFA and buffer volumes by the total number of samples that will be stained to get the total volume needed from each reagent. Add 5 mL of Post-Staining Fixing Solution to each well.
Place the coverslips in the Post-Staining Fixing Solution (PS) wells.
Incubate for 00:10:00 at Room temperature .
10m
Prepare ice-cold methanol during incubation.
- Place a 6-well TC plate on ice.
- Pipet methanol up and down to equilibrate the serological pipet tip.
- Add 5 mL methanol to one well per sample.
Remove samples from Post-Staining Fixing Solution (PS) and rinse the samples by dunking the coverslips 2-3 times each in the 1x DPBS (PBS) wells for a total of 3 washes.
Remove the coverslips from 1x DPBS (PBS) and place in ice-cold methanol.
Incubate for 00:05:00 .
5m
Bring plate containing 1x DPBS (PBS) to the ice bucket for quick transfer from methanol.
After incubation, rinse the samples by dunking the coverslips 2-3 times each in the 1x DPBS (PBS) wells for a total of 3 washes.
Rinse the humidity chamber if not previously washed.
Prepare the Final Fixative Solution. For 1-5 samples, mix the following:
1000 µL 1x DPBS + 20 µL CODEX Fixative Reagent
*20 μL of CODEX Fixative Reagent is one aliquot, thaw and spin down right before use.
Vortex gently to mix.
Remove coverslips from wells and place them in the humidity chamber.
Add Room temperature of Final Fixative Solution to the top corner of each sample.
Incubate for 00:20:00 .
20m
Remove samples from the humidity chamber and rinse the samples by dunking the coverslips 2-3 times each in the 1x DPBS (PBS) wells for a total of 3 washes.
Place the coverslips in a new TC plate with 5 mL Storage Buffer (SB) per sample in each well.
STOPPING POINT- use samples directly to run a CODEX experiment or store up to two weeks at 4C.
CODEX-tagged Dyes Cycle Prep
CODEX-tagged Dyes Cycle Prep
Gather necessary materials:
Note
Materials needed:
- 96-well plates (CODEX Mounting Kit 1.0 @ RT)
- 96-well plate foil seal (CODEX Mounting Kit 1.0 @ RT)
- 10X CODEX Buffer (CODEX Assay Kit 1.0 @ RT)
- CODEX Assay Reagent (CODEX Assay Kit 1.0 @ -20C)
- CODEX Nuclear Stain (CODEX Assay Kit 1.0 @ -20C)
- CODEX-Tagged Dyes (CODEX Antibody Kit 1.0 and CODEX Conjugation Kit 1.0 @ -20C)
- Nuclease-free water
- 1.5 mL amber Eppendorf tubes
Determine the CODEX-tagged dyes combination for each running cycle. Each cycle may contain up to one HOECHST/DAPI dye, one FAM/FITC dye, one Cy3/TRITC dye, and one Cy5 dye.
Label an amber Eppendorf tube for each CODEX-tagged dyes combination.
Prepare the stock solution of CODEX Reporter Stock Solution.
For 5 wells (cycles) per plate, mix the following:
150 µL 10x CODEX Buffer + 125 µL Assay Reagent + 5 µL Nuclear Stain
+ 1220 µL Nuclease-free water
Double or triple the volume of each reagent if running 10 or 15 cycles per plate. Mix solution by gently inverting.
Add CODEX Reporter Stock Solution to each labeled amber Eppendorf tube.
| A | B | C | D | |
| 3 CODEX-tagged dyes | 2 CODEX-tagged dyes | 1 CODEX-tagged dye | ||
| CODEX Reporter Stock Solution Volume per tube | 235 μL | 240 μL | 245 μL |
Thaw each CODEX-tagged dye immediately before use.
Quickly spin down the CODEX-tagged dyes.
Add 5 µL of each CODEX-tagged dye to its corresponding tube.
Mix contents of each tube by gently pipetting.
Transfer 245 µL of solution from each tube to its designated well in the 96-well plate.
Prepare a strip of foil seal and cover all wells containing CODEX-tagged dyes.
STOPPING POINT- use dyes directly to run a CODEX experiment or store up to two weeks at 4C.
Running the CODEX instrument
Running the CODEX instrument
20m
20m
Gather necessary materials.
Note
Materials needed:
- 96-well plate foil seal (CODEX Mounting Kit 1.0 @ RT)
- 2 CODEX Gaskets (CODEX Mounting Kit 1.0 @ RT)
- 10X CODEX Buffer (CODEX Assay Kit 1.0 @ RT)
- CODEX Nuclear Stain (CODEX Assay Kit 1.0 @ -20C)
- Stage Insert with Lid (CODEX Instrument)
- 600-2000 mL glass beakers
- Bent-tip tweezers
- 1.5 mL and 2 mL Eppendorf tubes
- Milli-Q water
- DMSO
- Aspirator setup
- Pipette tip box lid
- Ice Bucket
Turn on the CODEX Instrument.
Remove the antibody-stained tissue section(s) adhered to the Poly-lysine coated coverslip and the sealed, pre-loaded 96-well plate containing the Reporter Master Mix solutions from 4C. Allow them to equilibrate to Room temperature for at least 00:15:00 .
15m
Prepare 1X CODEX Assay Buffers in 600-2000 mL glass beakers.
| A | B | C | D | E | F | G | H | I | |
| 5 CODEX cycles | 8 CODEX cycles | 10 CODEX cycles | 12 CODEX cycles | 15 CODEX cycles | 18 CODEX cycles | 20 CODEX cycles | 25 CODEX cycles | ||
| 10X CODEX Buffer | 31 mL | 44 mL | 53.3 mL | 63.3 mL | 78.5 mL | 93.5 mL | 103.5 mL | 128.5 mL | |
| Milli-Q water | 281 mL | 394 mL | 480.5 mL | 570.5 mL | 705.5 mL | 840.5 mL | 930.5 mL | 1155.5 mL | |
| Total | 312 mL | 438 mL | 534 mL | 634 mL | 784 mL | 934 mL | 1034 mL | 1284 mL | |
| DMSO | 236 mL | 318 mL | 386 mL | 454 mL | 556 mL | 658 mL | 726 mL | 896 mL |
1X CODEX Buffer
Set aside 7 mL of 1x CODEX Buffer in a 15 mL conical tube for later use.
Place generated 1x CODEX Buffer in the CODEX Buffer Bottle, labeled Bottle One and DMSO in the amber bottle, labeled Bottle Two.
Load the stage insert.
Untwist the knobs at the top of the stage insert to remove the lid with the fluidic lines.
Remove the old coverslip using the forceps, push the corners of the gaskets towards the center to release the seal between the coverslip and the gasket.
Gently remove the coverslip using the forceps.
If the coverslip breaks, remove all glass pieces and rinse with water.
Soak fresh gaskets in 1X CODEX Buffer or in Storage Buffer for a few seconds.
Place the first gasket inside the squared well at the center of the stage insert.
Gently tap with forceps to ensure it adheres to the stage insert surface.
Gently place the tissue sample on top of the gasket, making sure it is perfectly inserted and the tissue is facing upwards.
Quickly and gently tap the coverslip edges with the forceps to ensure it adheres to the gasket.
Quickly place the second gasket on top of the sample coverslip.
Quickly place the lid on top of the stage insert.
Secure the lid by turning the knobs.
Dispense 700 µL of 1X CODEX Buffer on the corner of the sample.
Remove any salts formed on the coverslip from drying using a kimwipe with Milli-Q water. Use a dry kimwipe to remove residual liquid.
Stain the CODEX sample with HOECHST.
Thaw CODEX Nuclear Staining Solution.
Have the 7 mL of 1x CODEX Buffer set aside previously within reach.
Prepare a 2 mL Eppendorf tube for the Nuclear Stain Solution.
Note
Prepare the Nuclear Stain Solution.
1999 µL 1x CODEX Buffer + 1 µL Nuclear Staining Solution
Remove the solution within the sample well.
Add 700 µL of Nuclear Stain Solution to the sample.
Incubate for 00:05:00 at Room temperature . Cover it from light.
5m
Remove the solution within the sample well.
Add 700 μL of 1x CODEX Buffer.
Repeat steps 83.7-83.8 for a total of 5 washes.
Add 700 µL of 1x CODEX Buffer.
Launch the CODEX Instrument Management Software and enter cycle, marker id and exposure time information. Minimize the window.
Place the plate with the CODEX-tagged dyes in its designated place in the instrument.
Clean any dried buffer on the bottom of the coverslip with a wet kimwipe and dry off any residual liquid.
Turn on the Keyence scanner and load the cassette into the scanner.