TITLE: CODEX ® Multiplexed Imaging – tissue staining in TMA and whole tissue FFPE sections
AUTHORS: Anna Martinez Casals, Inna Sitnik, Christian M. Schürch, Mikael Malmqvist, Patrick E Macdonald, Emma Lundberg

[DESCRIPTION]
CODEX technology allows for highly multiplexed analysis of + 40 proteins on the same section relying on DNA-conjugated antibodies being commercialized by Akoya Biosciences (former name CODEX®, current name PhenoCycler™). This detailed protocol describes an adapted protocol, from CODEX User Manual Rev C , for tissue staining in Tissue MicroArrays (TMA) and whole tissue Formalin Fixed Paraffin Embedded (FFPE) sections to run into the CODEX system which is used in Emma Lundberg research group at Science for Life Laboratory;  KTH - Royal Institute of Technology.



[BEFORE_START]
Review the protocol before starting it to ensure having all the material needed.

[STEPS]
SECTION: Post staining steps
26. The day after: remove the coverslip from the humidity chamber, place the coverslip in a well with Staining Buffer (6-well plate) and immerse the coverslip 2-3 times. Incubate for 2 min. 


 


SECTION: Primary antibody staining steps
16. Using a coverslip forceps, transfer the coverslip into a well (6-well plate) with Hydration Buffer. Immerse the coverslip in the Hydration Buffer 2-3times.

 

 

SECTION: Tissue preparation: I. Coverslip coating &amp; sectioning
2. Carefully remove the poly-l-lysine, cover the coverslips with MiIli-Q water, stir slowly and let them settle during 30 s for a first wash. After the first wash, slowly remove the MiIli-Q water.
SECTION: Tissue preparation: I. Coverslip coating &amp; sectioning
1. Place coverslips in a glass beaker and cover them with  . Ensure all the coverslips are fully immersed in the solution avoiding overlapping, add a plastic wrap to prevent evaporation and let them coat  .

 

SECTION: Tissue preparation: I. Coverslip coating &amp; sectioning
4. Batch by batch, start transferring coverslips from the glass beaker to a sterile Petri dish with MiIli-Q water. Place 2 tissue wipes on the bench: take each individual coverslip and place it in a tissue wipe, once it is dried from one side move it to the next tissue wipe to dry the other side. Repeat the process with all the coverslips, changing the tissue wipes once they are wet.

 

SECTION: Tissue preparation: I. Coverslip coating &amp; sectioning
5. Place the completely dried coverslips in a new Petri plate to store them at Room temperature  till 2 months (label the Petri dish with the date). It is recommended that coverslips are coated with poly-L-lysine at least 2 days prior to tissue sectioning.
SECTION: Tissue preparation: I. Coverslip coating &amp; sectioning
6. Section the TMA or whole tissue block on the coated coverslips, trying to center it as much as possible. Recommended   /section.
SECTION: Tissue preparation: I. Coverslip coating &amp; sectioning
3. Repeat the  .
SECTION: Tissue preparation
None. Meanwhile prepare the pressure cooker, to perform antigen retrieval step, filling it with ddH2O using the following settings: 106-110 °C and low pressure allowing to heat up.
 

SECTION: Tissue preparation: II. Tissue treatment
7. Place the coverslip (tissue facing up, see Fig. 1) in a slide warmer and bake it at 55 °C 
 during 60 min 

  
 

SECTION: Tissue preparation: II. Tissue treatment
9. Start the deparaffinization and hydration steps (Fig. 2): place the coverslip staining rack carefully in each of the next solvents, following the order, during 5 min. Ensure to close the lid of each of the containers to avoid evaporation.

 

 
SECTION: Tissue preparation: II. Tissue treatment
9.1.  
SECTION: Tissue preparation: II. Tissue treatment
9.2.   
SECTION: Tissue preparation: II. Tissue treatment
9.3.   
SECTION: Tissue preparation: II. Tissue treatment
9.4.   
SECTION: Tissue preparation: II. Tissue treatment
9.5. 90% Ethanol prepared with ddH2O.
SECTION: Tissue preparation: II. Tissue treatment
9.6. Meanwhile prepare the pressure cooker, to perform antigen retrieval step, filling it with ddH2O using the following settings: 106-110 °C and low pressure allowing to heat up.

 


SECTION: Tissue preparation: II. Tissue treatment
9.7. 70% Ethanol prepared with ddH2O.
SECTION: Tissue preparation: II. Tissue treatment
9.8. 50% Ethanol prepared with ddH2O.
SECTION: Tissue preparation: II. Tissue treatment
9.9. 30% Ethanol prepared with ddH2O.
SECTION: Tissue preparation: II. Tissue treatment
9.10. ddH2O.
SECTION: Tissue preparation: II. Tissue treatment
9.11. ddH2O.
SECTION: Tissue preparation: II. Tissue treatment
9.12. Prepare the 1x citrate buffer solution in the container from the heat retrieval system: 25 ml    + 225 ml ddH2O.
SECTION: Tissue preparation: II. Tissue treatment
9.13. Transfer the coverslip staining rack to the container with the 1x citrate buffer and place the lid on top.
SECTION: Tissue preparation: II. Tissue treatment
9.14. Place the covered container into the heat retrieval system and set up the following settings: 114-121 °C, high pressure for 20 min.
SECTION: Tissue preparation: II. Tissue treatment
9.15. Remove the container from the heat retrieval system and allow to equilibrate to RT for at least 30min (otherwise tissue detachment may occur on the slide).
SECTION: Tissue preparation: II. Tissue treatment
9.16. Transfer the coverslip staining rack into a container with ddH2O and leave it for 2 min then transfer it to a second container with ddH2O for additional 2 min.
SECTION: Optional: Photobleaching treatment
10. 
 

Use two LED-lights to apply directly to the tissue to reduce the tissue autofluorescence. To avoid direct exposure to the lights, use a container (Fig. 3) and place inside the LED-lights (Fig. 4) creating a sandwich where the sample will be located between them in a falcon tube.

  
 


 
SECTION: Optional: Photobleaching treatment
11. Prepare the photobleaching solution in a 50 ml tube: 25 ml 1x PBS + 4.5 ml 30% (w/w)   and 0.8 ml 1M NaOH.
SECTION: Optional: Photobleaching treatment
12. Transfer the coverslip staining rack with the sample into the 50 ml tube containing the solution and place the tube in the rack between the LED-lights. Turn them on at maximum capacity during 45 min.
SECTION: Optional: Photobleaching treatment
13. Depending of the type of tissue, the sample may undergo a second photobleaching incubation repeating   and  .

SECTION: Optional: Photobleaching treatment
14. Wash the sample with 1x PBS during 3 min.
SECTION: Optional: Photobleaching treatment
15. Repeat the     for a total of 4 washes.
SECTION: Primary antibody staining steps
17. Let the coverslip sit for 5 s and transfer the coverslip to a second well with Hydration Buffer, let it sit for 5 s.

 


SECTION: Primary antibody staining steps
18. Transfer the coverslip to the well containing the Staining Buffer and allow the sample to equilibrate for 20-30min (do not exceed the 30min).

 

SECTION: Primary antibody staining steps
21. Create an humidity chamber: fill the stainTray slide staining system (Fig. 5) with ddH2O to create a humidity environment to incubate the tissue with the 1ary antibodies.

 


 
SECTION: Primary antibody staining steps
22. Cut a rectangular piece of parafilm, roughly the size and shape of the non-label portion of a microscope slide.
SECTION: Primary antibody staining steps
23. Take a new microscope slide and place it in the stainTray slide staining system. Add the parafilm on top of the slide.
SECTION: Primary antibody staining steps
24. Add the total volume of Antibody Cocktail solution (50µl) in a drop on top of the parafilm and add the coverslip face down (the tissue is in contact with the solution). Close the humidity chamber.
SECTION: Primary antibody staining steps
25. Incubate at  4 °C  .

SECTION: Post staining steps
27. Transfer the sample to a second well with Staining Buffer and incubate the samples for additional 2 min.

 


SECTION: Post staining steps
28. Prepare the Post-Staining Fixing Solution:  500 µl   + 4500 µl Storage Buffer and add 5 ml of the solution in a well (new 6-well plate).

SECTION: Post staining steps
29. Transfer the sample to the well containing the Post-Staining Fixing Solution and incubate at Room temperature  for 10 min.


 

SECTION: Post staining steps
30. Meanwhile, add 5mL of cold methanol   into a well (new 6-well plate) and place the plate on an on ice bucket (Fig. 6).

  
 

SECTION: Post staining steps
31. Remove the coverslip from the Post-Staining Fixing Solution and place it in a well containing 1x PBS. Immerse it 2-3 times. 

 

SECTION: Post staining steps
32. Repeat    for a total of 3 washes.

 

SECTION: Post staining steps
33. Remove the coverslip from the last 1x PBS and transfer it into the ice-cold methanol well. Incubate on on ice  for 5 min.

 

SECTION: Post staining steps
34. After the incubation, immediately transfer the coverslip to a well with fresh 1x PBS. Immerse the sample 2-3 times.  

 

SECTION: Post staining steps
35. Repeat   for a total of 3 washes.

 


SECTION: Post staining steps
36. Rinse and dry the StainTray slide staining system.
SECTION: Post staining steps
37. Prepare Final Fixative Solution: 1 ml 1x PBS + 20 µl of CODEX Fixative Reagent tube (do not re-freeze again) and vortex it. 
SECTION: Post staining steps
38. Take a new microscope slide and place it in the stainTray slide staining system. Add a parafilm piece on top of the slide.
SECTION: Post staining steps
39. Pipette 200 µl of Final Fixative Solution on top of the parafilm and add the coverslip face down (the tissue is in contact with the fixative). Incubate for 20 min.

SECTION: Post staining steps
40. Remove the coverslip from the stainTray slide staining system and transfer it to a first well of 1x PBS. Immerse the sample 2-3 times.  

 

SECTION: Post staining steps
41. Repeat   for a total of 3 washes.

 

SECTION: Post staining steps
42. Label a new 6-well plate and add 5 ml of Storage Buffer into a well: transfer the coverslip into the Storage Buffer with the tissue facing up.

 

SECTION: Post staining steps
43. Seal the 6-well plate and store it at 4 °C.

 

SECTION: Tissue preparation: II. Tissue treatment
8. Transfer the coverslip to a coverslip staining rack and let it cool down for 5 min.

 


SECTION: Primary antibody staining steps
19. Prepare the CODEX Blocking Buffer using the following components and volumes:

 

SECTION: Primary antibody staining steps
20. Prepare the Antibody Cocktail solution diluting the pre-conjugated 1ary antibodies in CODEX Blocking Buffer (final volume = 50µl). Pipette gently.