TITLE: CODEX FFPE Staining and Fixation  
AUTHORS: wbei

[DESCRIPTION]
Detailed protocol for preparing, staining, and fixing FFPE slides for use with Akoya flowcells in the Akoya phenocycler (CODEX). Slides are ready to be used with the Akoya phenocycler and the Akoya protocol following this protocol.

[STEPS]
SECTION: Staining
1. Bake slides at 70 °C in an oven/incubator

 

SECTION: Staining
2. Deparaffinize and rehydrate the slides


SECTION: Staining
2.1. Incubate slides for 21 min in xylene in a coplin jar

SECTION: Staining
2.2. Place slides in ST4020 Linear Staining vial and start the staining protocol
SECTION: Staining
2.3. Each step is 3 minutes
Xylene x3 -> 100% EtOH x2 -> 95% EtOH x2 -> 80% EtOH x1 -> 70% EtOH x1 -> ddH2O x3

 


SECTION: Staining
2.4. After starting the linear staining step, fill slide chamber with HIER 1X buffer and incubate HIER buffer at 75 °C  (170 °F) in pressure cooker filled with enough water (cover chamber with aluminum foil)

SECTION: Staining
2.5. Transfer slides to chamber containing heated HIER buffer
SECTION: Staining
2.6. Put the chamber back in the pressure cooker, heat to 97 °C (205 °F) and incubate for 1050 s  min 
 


SECTION: Staining
2.7. Stop the pressure cooker and turn it off, leave the chamber in the water bath for 20 min  to cool down slowly.
SECTION: Staining
2.8. Take the chamber out of pressure cooker, cool down at RT for about 30 min until around RT

SECTION: Staining
3. Wash tissue
SECTION: Staining
3.1. Place slide in coplin jar containing 80 mL of 1X TBS IHC Wash Buffer with Tween 20
(https://www.cellmarque.com/ancillaries/CM/2087/TBS-IHC-Wash-Buffer-Tween-20)
SECTION: Staining
3.2. Incubate on a shaker for 10 min at around 100 rpm

SECTION: Staining
4. Make blocking buffer solution (amount depends on sample area. ~120-140 uL/slide)
1 mL = 780 uL of S2 (RT), 50 uL of B1, 50 uL of B2, 50 uL of B3, 70 uL of BC4
NOTE: can store remaining buffer at 4°C 
SECTION: Staining
5. Block
SECTION: Staining
5.1. Tap off excess wash buffer, wipe edges and back with Kim Wipes, and place slides in humidity chamber (or use pipette box with water and paper towel underneath)
SECTION: Staining
5.2. Add 120-140 ul (depending on the sample area) of blocking buffer and incubate for 60 min   at RT in humidity chamber
(Can leave much longer and just need to add solution so tissue does not dry out)

SECTION: Staining
6. Dilute antibodies in blocking buffer to a total of 120 ul (ratio of blocking buffer: antibody cocktails should be >= 1 v/v).
SECTION: Staining
7. Antibody Staining
SECTION: Staining
7.1. After 1 hour of blocking, tap off excess buffer and add 120 ul of conjugated antibody solution. Cover tissue area with Parafilm

SECTION: Staining
7.2. Incubate 30 min  at 4 °C in humidity chamber on a shaker
SECTION: Fixation
8. Wash tissue. 
SECTION: Fixation
8.1. Place slide in chamber containing S2 buffer. 
SECTION: Fixation
8.2. Incubate for 4 min on a shaker

SECTION: Fixation
9. Fix tissue. Prepare 1.6% PFA (dilute from 16% PFA) solution in S4 buffer (1:10 (v/v)). NOTE: Use fresh vial of PFA every 1-2 weeks.  
SECTION: Fixation
9.1. Place slide in humidity chamber and add 100 uL of PFA solution or enough to cover the tissue
SECTION: Fixation
9.2. Incubate for 10 min. 

SECTION: Fixation
10. Wash tissue. 
SECTION: Fixation
10.1. Place slide in chamber containing 1x PBS for 1 min on a shaker

SECTION: Fixation
11. Ice-cold methanol incubation. Place slide chamber on an ice and fill with cold methanol (4 °C). 

SECTION: Fixation
11.1. Remove slide from chamber containing 1x PBS and place in chamber containing ice-cold methanol. 
SECTION: Fixation
11.2. Incubate for 5 min. 

SECTION: Fixation
12. Wash tissue. 
SECTION: Fixation
12.1. Remove slide from cold methanol and place in chamber containing 1x PBS (ok to use same PBS as from step #10). 
SECTION: Fixation
12.2. Incubate for 1 min on a shaker

SECTION: Fixation
13. Fix tissue. Prepare final fixative solution. Remove FIX aliquot from -20°C freezer right before use and let it melt. Add entire contents (~20 ul) to 1 ml of 1x PBS. Mix fully. 
SECTION: Fixation
13.1. Add 100 uL of fixative solution (or enough to cover the tissue), taking care not to pipette directly onto tissue. 
SECTION: Fixation
13.2. Incubate for 20 min in a humidity chamber.

SECTION: Fixation
14. Wash tissue. 
SECTION: Fixation
14.1. Place slide in chamber containing 1x PBS for 1 min on a shaker

SECTION: Fixation
15. Assemble the Akoya flowcells to the slides directly or store slides in S4 buffer, and assemble later.