TITLE: CODEX Sample Preparation and Staining Experiment Protocol
AUTHORS: Kavya.Anjani  , Miguel.Rivera  , Zoltanlaszik  

[DESCRIPTION]
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.

[STEPS]
SECTION: Tissue Sample Preparation Prior to CODEX Staining
1. Gather necessary materials
 

SECTION: Tissue Sample Preparation Prior to CODEX Staining
2. Coat coverslips with poly-lysine.
 

SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.1. Gently place 20 coverslips on the bottom of the glass beaker and slowly swirl the beaker to fan out the coverslips.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.2. Add 70 mL of poly-lysine solution to the beaker, making sure to submerge all coverslips.

SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.3. To prevent evaporation, use the saran wrap to cover the beaker and seal the wrap with a rubber band.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.4. STOPPING POINT- Incubate coverslips in poly-lysine solution for a minimum of 720 min and up to one week at Room temperature.

SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.5. After incubation, dispose of the poly-lysine solution into a proper waste container.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.6. Fill the beaker halfway with Milli-Q water and swirl contents to rinse coverslips.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.7. Let the beaker and coverslips sit for 1 min.

SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.8. Dispose of the water into the sink.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.9. Repeat steps 2.5-2.8 for a total of 7 washes.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.10. Fill the beaker halfway with Milli-Q water.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.11. Place two sets of paper towels on the benchtop.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.12. Remove the coverslips from the beaker, then separate and lay each piece face down on the first set of paper towels.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.13. Invert each coverslip to dry the reverse side on the second set of paper towels.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
2.14. Once the coverslips dry completely, collect them in a plastic petri dish to store for up to 2 months.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
3. Cut and embed frozen tissues on poly-lysine coated coverslips.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
3.1. Place poly-lysine coated coverslips in the cryostat chamber to equilibrate.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
3.2. Cut frozen tissue slices with   -    thickness.
 



SECTION: Tissue Sample Preparation Prior to CODEX Staining
3.3. Gently place tissue slice in the center of the coverslip.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
3.4. 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.
SECTION: Tissue Sample Preparation Prior to CODEX Staining
3.5. Store tissue slices separated at-80 °C for up to 6 months prior to staining.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
4. Gather necessary materials.
 

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
5. Calculate and obtain 50 µg of each antibody that will be conjugated.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
6. Label a 50kDA MWCO filter unit (both the filter device and the collection tube) for each antibody that will be conjugated.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
7. Add 500 µL of Filter Block Solution to the filter device.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
8. Spin down the filter unit at 12000 x g, 2 min.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
9. Discard all liquid (from both the filter device and the collection tube).
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
10. Add 50 µg of antibody to the filter device.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
11. Spin down the filter unit at 12000 x g, 8 min.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
12. Prepare the Antibody Reduction Master Mix while spinning.
 

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
13. Discard flow through.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
14. Add 260 µL of Antibody Reduction Master Mix to each filter device.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
15. Vortex solution in filter device for2 s - 3 s.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
16. Incubate tube for 30 min at -80 Room temperature.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
17. Spin down the filter unit at 12000 x g, 8 min.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
18. Discard flow-through.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
19. Add 450 µL of Antibody Conjugation Solution to each filter device.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
20. Spin down the filter unit at 12000 x g, 8 min.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
21. Prepare the CODEX antibody tag solution while spinning.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
21.1. Label a new tube for each CODEX antibody tag (one tag per antibody).
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
21.2. Add 210 µL of Antibody Conjugation Solution to each tube in step a. Set aside.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
21.3. Add 10 µL of Milli-Q water to resuspend each CODEX antibody tag (retrieved from -20 just prior). Mix by pipetting.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
21.4. Add the entire resuspended CODEX antibody tag to step b and mix by pipetting.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
22. Discard flow-through.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
23. Add the CODEX antibody tag solution prepared in step 21 to the corresponding filter device.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
24. Close the lid and vortex the filter unit for 2-3 sec.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
25. Incubate tube for 120 min at -80 Room temperature.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
26. After 120 min, set aside 5 µL of the purified solution for QC.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
27. Spin down the filter unit at 12000 x g, 8 min.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
28. Discard flow-through.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
29. Add 450 µL of Antibody Purification Solution to each filter device.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
30. Spin down the filter unit at 12000 x g, 8 min.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
31. Discard flow-through.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
32. Repeat steps 29-31 two more times for a total of three times.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
33. There will be solution left in the filter device.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
34. Label a new collection tube for each filter device.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
35. Add 100 µL Antibody Storage Solution to each filter device.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
36. Invert the filter device into its newly labeled collection tube.
SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
37. Spin down the filter unit at3000 x g, 2 min.

SECTION: Antibody Conjugation with CODEX-tags for Custom Panel
38. Transfer solution in the collection tube to a labeled 2 mL screw-top tube and store at 4 °C for up to 1 year.

SECTION: Tissue Staining
39. Gather necessary materials.
 

SECTION: Tissue Staining
40. Prepare a humidity chamber.
SECTION: Tissue Staining
40.1. Place wet paper towels at the bottom of an empty pipet tip box. 
SECTION: Tissue Staining
40.2. Fill the box with enough water to cover the towels completely.
SECTION: Tissue Staining
40.3. Recover the pipet tip tray and lid after rinsing.
SECTION: Tissue Staining
41. Prepare Drierite absorbent beads.
SECTION: Tissue Staining
41.1. Fill an empty pipet tip box with Drierite beads (about 1~2 cm deep).
SECTION: Tissue Staining
42. Prepare the following plate configuration for CODEX solutions:
  

SECTION: Tissue Staining
43. Prepare 10 mL of acetone in a 50 mL beaker for each tissue.

SECTION: Tissue Staining
44. Remove the tissue sections from -80C and place the coverslips on Drierite absorbent beads.
SECTION: Tissue Staining
45. Let the tissue sections dry for 2 min at 4 Room temperature.

SECTION: Tissue Staining
46. Place the tissue samples face-up in the beakers containing acetone.
SECTION: Tissue Staining
47. Incubate for 10 min.

SECTION: Tissue Staining
48. Remove the coverslips from acetone and place them in the humidity chamber to dry for 2 min at 4 Room temperature.

SECTION: Tissue Staining
49. Hydrate tissue samples following the plate configuration.
SECTION: Tissue Staining
49.1. Place the coverslips in the Hydration Buffer wells (dip 2-3 times before fully submerging the slide). Ensure tissue is facing up.
SECTION: Tissue Staining
49.2. Incubate for 2 min.

SECTION: Tissue Staining
49.3. Place the coverslips in the next set of Hydration Buffer wells.
SECTION: Tissue Staining
49.4. Incubate for2 min.

SECTION: Tissue Staining
49.5. Prepare Pre-Staining Fixing Solution while incubating tissue.
 

SECTION: Tissue Staining
49.6. Place the coverslips in the Pre-Staining Fixing Solution wells.
SECTION: Tissue Staining
49.7. Incubate for 10 min at 4 Room temperature.

SECTION: Tissue Staining
49.8. 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.
SECTION: Tissue Staining
49.9. Place the samples in the Staining Buffer wells and let equilibrate for 20 min - 30 min.

SECTION: Tissue Staining
50. Prepare the antibody cocktail during equilibration.
SECTION: Tissue Staining
50.1. 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.
SECTION: Tissue Staining
50.2. Calculate the total antibody volume per antibody cocktail.

Total Ab volume = (# of antibodies) x (# of samples) x (Ab volume/sample)]
 

SECTION: Tissue Staining
50.3. 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
 

SECTION: Tissue Staining
50.4. Combine the antibodies and the CODEX Blocking Buffer to make 210 µL Antibody Cocktail Solution/tissue sample.

SECTION: Tissue Staining
51. Remove the coverslips from Staining Buffer and place in the humidity chamber.
SECTION: Tissue Staining
52. Quickly add 200 µL of Antibody Cocktail Solution to each sample from the top corner of the coverslip.

SECTION: Tissue Staining
53. Place lid back on the humidity chamber.
SECTION: Tissue Staining
54. Incubate for 180 min at 4 Room temperature.

SECTION: Tissue Staining
55. Prepare the following plate configuration for CODEX solutions ~1 hour before the 3-hour incubation ends:  

SECTION: Tissue Staining
56. Wash the tissue samples following the plate configuration.
SECTION: Tissue Staining
56.1. Place the coverslips in the Staining Buffer (SB) wells (dip 2-3 times before fully submerging the slide). Ensure tissue is facing up.
SECTION: Tissue Staining
56.2. Incubate for 2 min.

SECTION: Tissue Staining
56.3. Place the coverslips in the next set of Staining Buffer (SB) wells.
SECTION: Tissue Staining
56.4. Incubate for 2 min.

SECTION: Tissue Staining
56.5. 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.

SECTION: Tissue Staining
56.6. Place the coverslips in the Post-Staining Fixing Solution (PS) wells.
SECTION: Tissue Staining
56.7. Incubate for 10 min at 4 Room temperature.

SECTION: Tissue Staining
56.8. 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.
SECTION: Tissue Staining
56.9. 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.
SECTION: Tissue Staining
56.10. Remove the coverslips from 1x DPBS (PBS) and place in ice-cold methanol.
SECTION: Tissue Staining
56.11. Incubate for 5 min.

SECTION: Tissue Staining
56.12. Bring plate containing 1x DPBS (PBS) to the ice bucket for quick transfer from methanol.
SECTION: Tissue Staining
56.13. 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.
SECTION: Tissue Staining
57. Rinse the humidity chamber if not previously washed.
SECTION: Tissue Staining
58. 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.
SECTION: Tissue Staining
59. Remove coverslips from wells and place them in the humidity chamber.
SECTION: Tissue Staining
60. Add 4 Room temperature of Final Fixative Solution to the top corner of each sample.

SECTION: Tissue Staining
61. Incubate for 20 min.

SECTION: Tissue Staining
62. 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.
SECTION: Tissue Staining
63. Place the coverslips in a new TC plate with 5 mL Storage Buffer (SB) per sample in each well.

SECTION: Tissue Staining
64. STOPPING POINT- use samples directly to run a CODEX experiment or store up to two weeks at 4C.
SECTION: CODEX-tagged Dyes Cycle Prep
65. Gather necessary materials:
 

SECTION: CODEX-tagged Dyes Cycle Prep
66. 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.
SECTION: CODEX-tagged Dyes Cycle Prep
67. Label an amber Eppendorf tube for each CODEX-tagged dyes combination.
SECTION: CODEX-tagged Dyes Cycle Prep
68. 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.
SECTION: CODEX-tagged Dyes Cycle Prep
69. Add CODEX Reporter Stock Solution to each labeled amber Eppendorf tube.
 

SECTION: CODEX-tagged Dyes Cycle Prep
70. Thaw each CODEX-tagged dye immediately before use.
SECTION: CODEX-tagged Dyes Cycle Prep
71. Quickly spin down the CODEX-tagged dyes.
SECTION: CODEX-tagged Dyes Cycle Prep
72. Add 5 µL of each CODEX-tagged dye to its corresponding tube.

SECTION: CODEX-tagged Dyes Cycle Prep
73. Mix contents of each tube by gently pipetting.
SECTION: CODEX-tagged Dyes Cycle Prep
74. Transfer 245 µL of solution from each tube to its designated well in the 96-well plate.

SECTION: CODEX-tagged Dyes Cycle Prep
75. Prepare a strip of foil seal and cover all wells containing CODEX-tagged dyes.
SECTION: CODEX-tagged Dyes Cycle Prep
76. STOPPING POINT- use dyes directly to run a CODEX experiment or store up to two weeks at 4C.
SECTION: Running the CODEX instrument
77. Gather necessary materials.
 

SECTION: Running the CODEX instrument
78. Turn on the CODEX Instrument.
SECTION: Running the CODEX instrument
79. 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 4 Room temperature for at least 15 min.

SECTION: Running the CODEX instrument
80. Prepare 1X CODEX Assay Buffers in 600-2000 mL glass beakers.

 
Set aside 7 mL of 1x CODEX Buffer in a 15 mL conical tube for later use.

SECTION: Running the CODEX instrument
81. Place generated 1x CODEX Buffer in the CODEX Buffer Bottle, labeled Bottle One and DMSO in the amber bottle, labeled Bottle Two.
SECTION: Running the CODEX instrument
82. Load the stage insert.
SECTION: Running the CODEX instrument
82.1. Untwist the knobs at the top of the stage insert to remove the lid with the fluidic lines.
SECTION: Running the CODEX instrument
82.2. 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.
SECTION: Running the CODEX instrument
82.3. Gently remove the coverslip using the forceps.
SECTION: Running the CODEX instrument
82.4. If the coverslip breaks, remove all glass pieces and rinse with water.
SECTION: Running the CODEX instrument
82.5. Soak fresh gaskets in 1X CODEX Buffer or in Storage Buffer for a few seconds.
SECTION: Running the CODEX instrument
82.6. Place the first gasket inside the squared well at the center of the stage insert.
SECTION: Running the CODEX instrument
82.7. Gently tap with forceps to ensure it adheres to the stage insert surface.
SECTION: Running the CODEX instrument
82.8. Gently place the tissue sample on top of the gasket, making sure it is perfectly inserted and the tissue is facing upwards.
SECTION: Running the CODEX instrument
82.9. Quickly and gently tap the coverslip edges with the forceps to ensure it adheres to the gasket.
SECTION: Running the CODEX instrument
82.10. Quickly place the second gasket on top of the sample coverslip.
SECTION: Running the CODEX instrument
82.11. Quickly place the lid on top of the stage insert.
SECTION: Running the CODEX instrument
82.12. Secure the lid by turning the knobs.
SECTION: Running the CODEX instrument
82.13. Dispense 700 µL of 1X CODEX Buffer on the corner of the sample.

SECTION: Running the CODEX instrument
82.14. Remove any salts formed on the coverslip from drying using a kimwipe with Milli-Q water. Use a dry kimwipe to remove residual liquid.
SECTION: Running the CODEX instrument
83. Stain the CODEX sample with HOECHST.
SECTION: Running the CODEX instrument
83.1. Thaw CODEX Nuclear Staining Solution.
SECTION: Running the CODEX instrument
83.2. Have the 7 mL of 1x CODEX Buffer set aside previously within reach.

SECTION: Running the CODEX instrument
83.3. Prepare a 2 mL Eppendorf tube for the Nuclear Stain Solution.
 


SECTION: Running the CODEX instrument
83.4. Remove the solution within the sample well.
SECTION: Running the CODEX instrument
83.5. Add 700 µL of Nuclear Stain Solution to the sample.

SECTION: Running the CODEX instrument
83.6. Incubate for 5 min at 4 Room temperature. Cover it from light.

SECTION: Running the CODEX instrument
83.7. Remove the solution within the sample well.
SECTION: Running the CODEX instrument
83.8. Add 700 μL of 1x CODEX Buffer.
SECTION: Running the CODEX instrument
83.9. Repeat steps 83.7-83.8 for a total of 5 washes.
SECTION: Running the CODEX instrument
83.10. Add 700 µL of 1x CODEX Buffer.

SECTION: Running the CODEX instrument
84. Launch the CODEX Instrument Management Software and enter cycle, marker id and exposure time information. Minimize the window.
SECTION: Running the CODEX instrument
85. Place the plate with the CODEX-tagged dyes in its designated place in the instrument.
SECTION: Running the CODEX instrument
86. Clean any dried buffer on the bottom of the coverslip with a wet kimwipe and dry off any residual liquid.
SECTION: Running the CODEX instrument
87. Turn on the Keyence scanner and load the cassette into the scanner.