Antibody Conjugation and CODEX Multiplexed Immunofluorescence of Fresh Frozen Tissue

Start with purified antibodies (no BSA, azides, glycerol, etc.). If BSA is present, remove with ab173231.

Block 50 kDa molecular weight cut off filters with 500µL of filter blocking solution.

Centrifuge at 12000x g,0h 0m 0s for 0h 2m 0s .

Dilute 50 µg of each antibody into 100µL of PBS and filter with 50 MW cut off filters.

Centrifuge at 12000x g,0h 0m 0s for 0h 8m 0s.

Reduce antibodies with the reduction mixture for 20-25 minutes at ~20°C.

Centrifuge the reduced antibodies at 12000x g,0h 0m 0s for 0h 8m 0s and add buffer solution.

Rehydrate oligonucleotide barcodes in 10µL nuclease free water and further dilute with 210µL of conjugation solution.

Respective barcode solutions are added to the reduced primary antibodies and incubated for 2h 0m 0s at ~ 20°C

*Purify the solution by buffer exchanging with 450µL purification solution. Centrifuge at 12000x g,0h 0m 0s for 0h 8m 0s . Discard flow-through.

twice.

Store in 100µL antibody storage solution at 4°C .

Remove fresh frozen tissue from -80°C and place inside a desiccator for 0h 20m 0s.

Submerge tissues in excess acetone for 0h 5m 0s.

Rehydrate tissues in hydration buffer for 0h 3m 0s inside a humidity chamber.

Fix the tissues for 0h 10m 0s in 1.6% paraformaldehyde in hydration buffer.

Submerge tissues in excess staining buffer for 0h 30m 0s.

Incubate tissues with a blocking buffer and primary antibody cocktail (1:200 antibody dilution) for 3h 0m 0s within a humidity chamber at ~ 20°C.

Rinse samples with staining buffer and fix with 1.6% paraformaldehyde in storage buffer for 0h 10m 0s.

Rinse with excess PBS and incubate in cold methanol for 0h 5m 0s. Rinse samples with excess PBS again.

Fix the samples with fixative solution for 0h 10m 0s before being stored in storage buffer at 4°C.

Dilute antibodies in reporter solution to a 1:200 dilution with 1µL of a 1mg/mL solution of DAPI or Hoechst 33342.

Fill respective bottles with nuclease free water, 1x CODEX buffer, and dimethyl sulfoxide.

Mount a blank coverslip into the coverslip microfluidic chamber and flush the fluidic lines.

Mount the prepared tissue sample and incubate with 1x CODEX buffer and 1µL of 1mg/mL of DAPI for 0h 5m 0s and wash with the automated CODEX system.

Set up tiled regions covering the tissue and focus at the tissue surface.

Perform automated microscopy using a Zeiss Axio equipped with a Colibri 7 LED light source and C13440 camera or other equivalent equipment.