TITLE: Protocol for CODEX Fixation Steps and Primary Antibody Staining for Induced Pluripotent Stem Cell-Derived Neurons
AUTHORS: Faria  Zafar, Alejandra  Torres, Laurin  Heinrich, Jasmine Singh, Cassandra Hempel, Oliver Braubach, Birgitt  Schuele

[DESCRIPTION]
CO-Detection by indEXing (CODEX) is a powerful technique to complement 'omics' technologies for in-depth phenotype analysis. CODEX allows the detection of 40+ targets in situ at single-cell resolution. 

This protocol describes the optimization of fixation and primary antibody staining of the human iPSC-derived neuronal cell cultures for CODEX. We show CODEX can be applied to iPSC neuronal cultures and developed fixation and staining protocols for the neurons to sustain the multiple wash-stain cycles of the technology without overt cell loss.

This protocol is adapted for the use of neuronal cell cultures from https://www.akoyabio.com/wp-content/uploads/2021/01/CODEX-User-Manual.pdf

[BEFORE_START]
Prepare and use biosafety cabinet chemical hood.



[GUIDELINES]
BSL-2 safety guidelines

[STEPS]
SECTION: A. Pre-staining first fixation (under the chemical hood)
2. Add an equal volume (1.5 mL) of 4% paraformaldehyde (PFA; prepare fresh 4% PFA with PBS from 32% PFA stock) for 5 min at room temperature (RT). The final PFA concentration is 2%.

SECTION: A. Pre-staining first fixation (under the chemical hood)
4. Add 3 mL 4% PFA to the well and incubate for 10 min at RT.

SECTION: A. Pre-staining first fixation (under the chemical hood)
5. Remove 4% PFA, wash the cells 2 times slowly with 3 mL PBS using a 1000ul pipette tip at RT.

SECTION: B. Pre-staining second fixation (under the chemical hood)
9. Place the coverslip on top of the Drierite beads with the sample facing up. Close the lid and wait for 2 min.  
Note: Make sure the sample is not visibly wet. If the sample coverslip is not completely dry after 2 min, place it in 10 mL of 100% acetone in a 50 mL beaker for 2 minutes, and again lay the coverslip on top of the Drierite beads for 2 minutes, then go to Step 10.
SECTION: B. Pre-staining second fixation (under the chemical hood)
10. Take the sample coverslip from the Drierite beads with tweezers and place the coverslip in 10 mL of 100% acetone in a 50 mL beaker and incubate for 5 min.       

SECTION: B. Pre-staining second fixation (under the chemical hood)
13. Wash with PBS 3 times3 min each at RT.

SECTION: B. Pre-staining second fixation (under the chemical hood)
14. Quench the sample on the coverslip for auto-fluorescence by placing them in 5 mL PBS in a separate 6-well plate and exposing them to a broad-spectrum LED light (DCenta A4 Ultra Bright LED Light Box Tracing Pad 25000Lux) at RT for 45 min. Repeat the step with fresh 5 mLPBS for 45 minagain.
  

SECTION: B. Pre-staining second fixation (under the chemical hood)
15. Then incubate the coverslip in Hydration Buffer 2 times for 3 min in 6-well plate from Step 6.

SECTION: C. Primary antibody staining
16. Note: Prepare Humidity Chamber before staining steps:
SECTION: C. Primary antibody staining
16.1. An empty pipette tip box with a lid and removable tray can be used as humidity chamber.

SECTION: C. Primary antibody staining
16.2. Wet a paper towel with ddH2O and place it at the bottom of the pipette box under the tray. 
SECTION: C. Primary antibody staining
16.3. Fill the pipette box with enough ddH2O at the bottom to fully cover the paper towel (approximately 1-2 cm deep). 
SECTION: C. Primary antibody staining
16.4. Rinse and dry the pipette tip tray before placing it back in the box.
SECTION: C. Primary antibody staining
16.5. Label different positions in the tray if working with multiple sample coverslips:
  

SECTION: C. Primary antibody staining
17. Before starting the staining, equilibrate the cells on the coverslips with 3 mL Staining Buffer at RT for up to 30 min in a new 6-well plate. 

SECTION: C. Primary antibody staining
18. Prepare CODEX®  blocking buffer as follows:
 
     CODEX Reagent      1 Sample      2 Samples       4 Samples      5 Samples      6 Samples        Staining   Buffer (ul)      181      362      724      905      1086        N   Blocker (ul)      4.75      9.5      19      23.75      28.5        G   Blocker (ul)      4.75      9.5      19      23.75      28.5        J   Blocker (ul)      4.75      9.5      19      23.75      28.5        S   Blocker (ul)      4.75      9.5      19      23.75      28.5        Total   (ul)      200      400      800      1000      1200   
 
SECTION: C. Primary antibody staining
19. Prepare primary antibody cocktail solution (depending on primary antibody dilution) with CODEX® blocking buffer, pipette to mix, or vortex gently. Briefly spin down the tube(s).

Example: 
Sample #: 1
Primary antibody #: 5
Each antibody dilution: 1/50 (4ul of each antibody for 200ul of the total solution)
Antibody cocktail solution: 20ul antibody mix X 180ul CODEX blocking buffer (from Step 18)

For further details please refer to p.48 (Understanding Antibody dilution) of the Akoya protocol: https://www.akoyabio.com/wp-content/uploads/2021/01/CODEX-User-Manual.pdf
SECTION: C. Primary antibody staining
20.  Aspirate up 190 µLof the antibody cocktail with a 200ul pipette tip. Set aside the tip with the pipettor.

SECTION: C. Primary antibody staining
21. Remove sample coverslip from the well-containing Staining Buffer with the tweezer and place the coverslip in the humidity tray chamber.
SECTION: C. Primary antibody staining
22. Quickly dispense the 190uL of the antibody cocktail to the top corner of the sample coverslip. Ensure that the liquid covers the entire tissue. 

Be careful not to pipette the solution directly on the tissue, and minimize bubbles!
SECTION: C. Primary antibody staining
23. Place the lid on the humidity chamber.
SECTION: C. Primary antibody staining
24. Incubate the samples in the humidity tray for 180 min at RT.

SECTION: D. Post-staining fixation
25. Prepare the following 6-well washing plate (needed after primary antibody incubation):
 
    

SECTION: D. Post-staining fixation
26. Take out the sample coverslip from the humidity chamber with tweezer, and place it in the upper left well with Staining Buffer (see Step 25). Lift and immerse sample coverslip 3 times to ensure the removal of the antibody cocktail from both sides of the coverslip.
SECTION: D. Post-staining fixation
27. Incubate for another 2 min.

SECTION: D. Post-staining fixation
28. Place sample coverslip in the second well (middle left, see pic in Step 25) containing Staining Buffer. Incubate for 2 min.

SECTION: D. Post-staining fixation
29. Place the sample coverslip in the well (lower left, see pic Step 25) containing Post-Staining Fixing Solution (1.6% PFA in storage buffer).
SECTION: D. Post-staining fixation
30. Incubate for 10 min at RT.

SECTION: D. Post-staining fixation
31. Place sample coverslip in the first well containing PBS. Lift and immerse the sample coverslip 2-3 times to ensure the Fixing Solution is removed from the top and bottom of the coverslip.
SECTION: D. Post-staining fixation
32. Quickly transfer the sample coverslip to the second well containing PBS. Lift and immerse the sample coverslip 2-3 times. Quickly transfer the sample coverslip to the third well containing PBS for a total of 3 washes.
SECTION: D. Post-staining fixation
33. Place a new 6-well plate on ice. Retrieve methanol from the refrigerator (4°C) and pipette methanol up and down 3 times to equilibrate the serological pipette tip to the cold temperature of methanol. Add 5 mL of cold (~4°C) methanol to one well per sample keeping the 6-well plate on ice.

SECTION: D. Post-staining fixation
34. Remove the sample coverslip from the well containing PBS and place them in the well containing ice-cold methanol.
SECTION: D. Post-staining fixation
35. Incubate on ice for 5 min.

SECTION: D. Post-staining fixation
36. Quickly transfer the sample coverslip from methanol to a newly prepared 6-well plate with 5 mLPBS in all wells at RT.

Note: Methanol dries tissue faster than other buffers. Move quickly to prevent sample damage.
SECTION: D. Post-staining fixation
37. Ensure the coverslip is fully immersed in PBS as methanol drying may cause the coverslip to float. Lift and immerse the sample coverslip 2-3 times to ensure methanol is removed from the bottom of the coverslip as well as the top.
SECTION: D. Post-staining fixation
38. Transfer the sample coverslip to the second PBS well. Lift and immerse the sample coverslip 2-3 times.
SECTION: D. Post-staining fixation
39. Transfer the sample coverslip to the third PBS well for a total of 3 washes. Lift and immerse the sample coverslip 2-3 times.
SECTION: D. Post-staining fixation
40. Prepare final fixative by adding 20 µLof BS3 (final fixative, thawed at RT) to 1000 µL of PBS. Mix gently by pipetting up and down. 

Note: Prepare fresh before each experiment.
SECTION: D. Post-staining fixation
41. Remove the sample coverslip from Step 41 and place it on the tray of the humidity chamber.
SECTION: D. Post-staining fixation
42. Add 200 µL of Final Fixative Solution to the top corner of the sample coverslip. Cover the entire section with reagent and remove all bubbles, if any, from the coverslip. Do not pipette the solution directly onto the tissue.

SECTION: D. Post-staining fixation
43. Place lid on humidity chamber and incubate for 20 min at RT.

SECTION: D. Post-staining fixation
44. Remove the sample coverslip from the humidity chamber and place it in a new well containing 5 mL PBS.

SECTION: D. Post-staining fixation
45. Lift and immerse the sample coverslip with tweezer 2-3 times to ensure Fixative Solution is removed.
SECTION: D. Post-staining fixation
46. Move the sample coverslip to the second well containing PBS. Lift and immerse the sample coverslip 2-3 times.
SECTION: D. Post-staining fixation
47. Move the sample coverslip to the third well containing PBS for a total of 3 washes. Lift and immerse the sample coverslip 2-3 times.
SECTION: D. Post-staining fixation
48. Label a new 6-well plate and pipette 5 mLof Storage Buffer and place the sample coverslip in the well with the cells facing up.

SECTION: D. Post-staining fixation
49. Seal the plate around the edges with parafilm.
SECTION: D. Post-staining fixation
50. Cells are ready for CODEX® imaging. Stained coverslip can be stored for 7 days at 4C before imaging.
SECTION: C. Primary antibody staining
16.6. Cover with the lid.
SECTION: A. Pre-staining first fixation (under the chemical hood)
1. For CODEX® imaging, iPSCs were plated and differentiated into neurons on specific 22x22 mm glass coverslips fitting the frame of the CODEX® instrument. Neurons were cultured on these coverslips in one well of a 6-well plate with 3ml of total media. 

On the day of fixation, remove 1.5 mL media from the well.

SECTION: A. Pre-staining first fixation (under the chemical hood)
3. Remove 2% PFA from the well with a 1000ul pipette tip.
SECTION: B. Pre-staining second fixation (under the chemical hood)
6. Prepare a 6-well plate with buffers as follows for further fixation steps and keep at RT:
   

SECTION: B. Pre-staining second fixation (under the chemical hood)
7. Prepare one well of a 6well plate with 1-2 cm thick Drierite beads.
SECTION: B. Pre-staining second fixation (under the chemical hood)
8. After Step 5, take out the sample coverslip from PBS using a bent-tip tweezer and tap dry from the corner with a Kimwipe.
SECTION: B. Pre-staining second fixation (under the chemical hood)
11. Place the sample coverslip on top of the Drierite beads with cells facing up (acetone evaporation) for 2 min at RT.

SECTION: B. Pre-staining second fixation (under the chemical hood)
12. Using the previously prepared 6-well plate from step 6, place the coverslip into 1.6% PFA/Hydration Buffer mix at RT for 10 min.