CODEX FFPE Staining and Fixation 

Bake slides at 70°C in an oven/incubator

Deparaffinize and rehydrate the slides

Incubate slides for 0h 21m 0s in xylene in a coplin jar

Place slides in ST4020 Linear Staining vial and start the staining protocol

Each step is 3 minutes

Xylene x3 -> 100% EtOH x2 -> 95% EtOH x2 -> 80% EtOH x1 -> 70% EtOH x1 -> ddH2O x3

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)

Transfer slides to chamber containing heated HIER buffer

Put the chamber back in the pressure cooker, heat to 97°C (205°F) and incubate for 0h 17m 30s min

Stop the pressure cooker and turn it off, leave the chamber in the water bath for 0h 20m 0s to cool down slowly.

Take the chamber out of pressure cooker, cool down at RT for about 0h 30m 0s until around RT

Wash tissue

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)

Incubate on a shaker for 0h 10m 0s at around 100 rpm

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

Block

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)

Add 120-140 ul (depending on the sample area) of blocking buffer and incubate for 1h 0m 0s at RT in humidity chamber

(Can leave much longer and just need to add solution so tissue does not dry out)

Dilute antibodies in blocking buffer to a total of 120 ul (ratio of blocking buffer: antibody cocktails should be >= 1 v/v).

Antibody Staining

After 1 hour of blocking, tap off excess buffer and add 120 ul of conjugated antibody solution. Cover tissue area with Parafilm

Incubate 0h 30m 0s at 4°C in humidity chamber on a shaker

Wash tissue .

Place slide in chamber containing S2 buffer.

Incubate for 0h 4m 0s on a shaker

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. 

Place slide in humidity chamber and add 100 uL of PFA solution or enough to cover the tissue

Incubate for 0h 10m 0s.

Wash tissue.

Place slide in chamber containing 1x PBS for 0h 1m 0s on a shaker

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

Remove slide from chamber containing 1x PBS and place in chamber containing ice-cold methanol.

Incubate for 0h 5m 0s.

Wash tissue .

Remove slide from cold methanol and place in chamber containing 1x PBS (ok to use same PBS as from step #10).

Incubate for 0h 1m 0s on a shaker

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.

Add 100 uL of fixative solution (or enough to cover the tissue), taking care not to pipette directly onto tissue.

Incubate for 0h 20m 0s in a humidity chamber.

Wash tissue .

Place slide in chamber containing 1x PBS for 0h 1m 0s on a shaker

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