TITLE: CODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation 
AUTHORS: Diane  Saunders, Conrad Reihsmann, Marcela Brissova, Alvin C. Powers

[DESCRIPTION]
This protocol describes the staining and pre-imaging preparation currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group prior to performing multiplexed imaging of the human pancreas using the CO-Detection by indEXing (CODEX®) platform (now PhenoCycler™; Akoya Biosciences). See also CODEX® Multiplexed Imaging | Modality overview.

[STEPS]
SECTION: Tissue Preparation
2. Remove coverslip from freezer and place face up in container with layer of  for 2 min to dry.
SECTION: Tissue Preparation
3. Place coverslip into an individual 10-mL beaker containing acetone, making sure entire tissue area is submerged. Incubate for 10 min.
SECTION: Tissue Preparation
4. Carefully remove coverslip from beaker and place back into Drierite container for another 2 min.
SECTION: Tissue Preparation
5. Transfer coverslip to 6-well plate and perform two washes in 3 mL Hydration Buffer, incubating for 2 min each time. If staining more than one sample at a time, make sure coverslip order/orientation in 6-well plates is kept consistent so you don't lose track of samples.
SECTION: Tissue Preparation
6. From second well of Hydration Buffer, transfer coverslip to well containing 3 mL Pre-Staining Fixation Solution (1.6 % (v/v) PFA in Hydration Buffer) and incubate for 10 min.
SECTION: Tissue Preparation
7. Wash coverslip twice in 3 mL Hydration Buffer to remove fixative. No incubation is required, and aliquots from step 5 can be reused.
SECTION: Tissue Preparation
8. Incubate coverslip in 3 mL Staining Buffer for 20 min. Place 6-well plate onto a fixed-angle rocker for the incubation period.
SECTION: Tissue Preparation
1. Prepare pre-staining reagents (per individual coverslip):

15-mL beaker with 10 mL acetone
 


2 wells Hydration Buffer
1 well Pre-staining Fixation Solution (1.6% PFA in Hydration Buffer)
1 well Staining Buffer
SECTION: Primary Antibody Stain
9. Prepare CODEX Blocking Buffer for primary antibody cocktail:

Staining Buffer       -    91 % (v/v)
N/G/J/S blockers  -    2.4 % (v/v) each

 

SECTION: Primary Antibody Stain
10. Calculate required amounts for each primary antibody (based on optimized dilution) as well as the total antibody volume. Remove the total volume from the Blocking Buffer, then add primary antibodies. Vortex gently.

ⓘ Preconjugated antibody dilutions: PhenoCycler Antibody Dilutions
SECTION: Primary Antibody Stain
11. Place coverslip face up in humidity chamber (can use an empty pipet tip box with damp paper towel in the bottom; coverslips rest on the removable insert). Carefully pipette 200 µL of antibody cocktail solution on top, making sure solution pools to cover the entire tissue area.


   
SECTION: Primary Antibody Stain
12. Incubate at Room temperature for 180 min.
SECTION: Post-Stain Procedure
14. Transfer coverslip from humidity chamber to 6-well plate and perform two washes in 3 mL Staining Buffer, incubating for 2 min each time.
SECTION: Post-Stain Procedure
15. From second well of Staining Buffer, transfer coverslip to well containing 3 mL Post-Staining Fixation Solution (1.6 % (v/v) PFA in Staining Buffer) and incubate for 10 min.
SECTION: Post-Stain Procedure
16. Wash coverslip three times in 3 mL DPBS to remove fixative. No incubation is required.
SECTION: Post-Stain Procedure
17. Incubate coverslip in 3 mL cold methanol on ice  for 5 min.
SECTION: Post-Stain Procedure
18. Wash coverslip three times in 3 mL DPBS to remove methanol. No incubation is required, and aliquots from step 16 can be reused.
SECTION: Post-Stain Procedure
20. Place coverslip face-up in humidity chamber and carefully pipette 200 µL of Final Fixative onto coverslip, making sure it covers the entire tissue area. Incubate at Room temperature for 20 min.

SECTION: Post-Stain Procedure
21. Wash coverslip three times in 3 mL DPBS to remove fixative. No incubation is required, and aliquots from steps 16/18 can be reused.
SECTION: Post-Stain Procedure
22. Transfer coverslip into 3 mL of Storage Buffer and label 6-well plate. Add a piece of parafilm before placing lid to ensure solution does not evaporate. Store at 4 °C for up to 2 weeks. Label the top of the plate with Sharpie or tape to keep track of sample(s).
SECTION: Post-Stain Procedure
13. Prepare post-staining reagents (per individual coverslip):
 

1 well Post-staining Fixation Solution (1.6% PFA in Staining Buffer)
2 wells Staining Buffer
1 well methanol on ice 
3 wells 1X DPBS
SECTION: Post-Stain Procedure
19. Prepare Final Fixative (2 % (v/v) Fixative Reagent in DPBS).

⚠ Thaw Fixative Reagent right before use and discard any unused volume. One tube provides adequate volume to fix up to 5 samples.
SECTION: Preparation of Reporter Plate
23. Gather supplies and reagents:

  
  
  
  


Prepare Reporter Stock Solution:

Nuclease-free water      -    81.33 % (v/v)
10X CODEX Buffer         -    10.00 % (v/v) 
Assay Reagent               -    8.33 % (v/v) 
CODEX Nuclear Stain    -    0.33 % (v/v) 

 

⚠ Ensure you prepare adequate Reporter Stock Solution to account for Blank cycles (no reporters); formulas in Table 2 reflect this.
SECTION: Preparation of Reporter Plate
24. Label one 1.5 mL light-blocking microcentrifuge tube for each reporter cycle and aliquot Reporter Stock Solution (250 µL per sample). You should have Reporter Stock Solution remaining (for Blank cycles) after aliquoting.
SECTION: Preparation of Reporter Plate
25. Prepare reporter mixes by adding appropriate reporters to microcentrifuge tubes (5 µL per sample for each reporter):

 

⚠ Make sure to vortex reporter tubes briefly before adding to reporter mix.
SECTION: Preparation of Reporter Plate
26. Mix contents of tubes by gently pipetting up and down with pipette or by brief vortex. Spin tubes using benchtop microcentrifuge to collect solution at bottom of tubes.
SECTION: Preparation of Reporter Plate
27. Add 250 µL of reporter mix to each corresponding well in a light-protected 96-well plate, making sure to leave blank wells before and after reporter mixes. Fill these wells (corresponding to Blank cycles) with 250 µL Reporter Stock Solution (no reporters in solution).


  
SECTION: Preparation of Reporter Plate
28. Seal plate with foil plate seal and store at 4 °C until imaging.