CODEX Reagent Setup

The following is an excerpt from Black et al., 2021, Nature Protocols

Black, S., Phillips, D., Hickey, J. W., Venkataraaman, V. G., Samusik, N., Goltsev, Y., Schürch, C. M., & Nolan, G. P. (2021). CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nature Protocols, 16(8), 3802-3835. https://doi.org/10.1038/s41596-021-00556-8

Reagent setup

PBS-Tween solution

Prepare a 0.1% (vol/vol) Tween solution in 1× DPBS. Store at room temperature (RT: 18–26 °C) for ≤6 months.

500 mM TCEP stock solution

Dissolve 717 mg of TCEP in 2.5 ml of ddH2O, adjust to pH 7.0 with sodium hydroxide and bring to a volume of 5 ml with ddH2O. Store at 4 °C for ≤1 year.

2.5 mM TCEP solution for antibody reduction

Add 5 μl of 500 mM TCEP and 5 μl of 500 mM EDTA (pH 8.0) to 990 μl of 1× DPBS. Mix by gently pipetting up and down. Prepare fresh before each experiment.

5 M NaCl solution

Dissolve 146.1 g of NaCl in 500 ml of ddH2O. Add 0.02 % (wt/vol) NaN3 to this solution. Store at RT for ≤1 year.

Buffer C

Add 1 ml of 1 M Tris (pH 7.0), 1 ml of 1 M Tris (pH 7.5), 30 ml of 5 M NaCl solution and 2 ml of 500 mM EDTA (pH 8.0) to 966 ml of ddH2O. Add 200 mg of NaN3 for a 0.02% (wt/vol) concentration in this solution. Store at RT for ≤1 year.

High-salt buffer C

Add 20 μl of 5 M NaCl solution to 380 μl of buffer C. Mix gently by pipetting up and down. Prepare fresh before each experiment.

High-salt PBS

Add45mlof5MNaClsolutionand25mlof10×DPBSto180mlofddH2O.Add50mgofNaN3 for a 0.02% (wt/vol) concentration in this solution. Store at RT for ≤1 year.

Stock antibody stabilizer solution

Add 0.02% (wt/vol) NaN3 to Candor PBS antibody stabilizer solution. Store at 4 °C for ≤1 year.

CODEX antibody stabilizer solution

Add 1 ml of 5 M NaCl solution and 100 μl of 500 mM EDTA (pH 8.0) to 9 ml of stock antibody stabilizer solution. Vortex briefly to mix. Store at 4 °C for ≤1 year.

Antigen retrieval solution, pH 9

Dilute Dako target retrieval solution at a 1:10 ratio with ddH2O (vol/vol). Prepare fresh before each experiment.

70% ethanol solution

Dilute 100% ethanol in ddH2O to make a 70% (vol/vol) solution. Store at RT for ≤3 months. 80% Ethanol

Dilute 100% ethanol in ddH2O to make a 80% (vol/vol) solution. Store at RT for ≤3 months. 95% Ethanol

Dilute 100% ethanol in ddH2O to make a 95% (vol/vol) solution. Store at RT for ≤3 months.

1× PBS for antigen retrieval

Add 150 μl of 10× PBS to 1.35 ml of ddH2O. Store at RT for ≤6 months.

TBS IHC wash buffer with Tween 20

Dilute 20× TBS IHC wash buffer with Tween 20 at a 1:20 ratio with ddH2O (vol/vol). Store at RT for ≤1 month.

CODEX staining buffer 1 (S1)

Add5mlof500mMEDTA(pH8.0),50mlof10×DPBS,2.5gofBSAand100mgofNaN3 to 445 ml of ddH2O. Gently mix. Store at 4 °C for ≤1 year.

1 M Na2HPO4

Dissolve 70.98 g of Na2HPO4 in 500 ml of ddH2O. Add 0.02% (wt/vol) NaN3 to this solution. Store at RT for ≤1 year.

1 M NaH2PO4

Dissolve 69 g of NaH2PO4•H2O in 500 ml of ddH2O. Add 0.02% (wt/vol) NaN3 to this solution. Store at RT for ≤1 year.

CODEX staining buffer 2 (S2)

Add250mlofS1,30.5mlof1MNa2HPO4,19.5mlof1MNaH2PO4 and25mlof5MNaCl solution to 175 ml of ddH2O. Adjust the pH to 6.8–7.0 with sodium hydroxide. Gently mix. Store at 4 °C for ≤1 year.

Blocking reagent 1 (B1)

Dissolve 10 mg of mouse IgG in 10 ml of S2. Store in 0.5-ml aliquots at 4 °C for ≤1 year. Blocking reagent 2 (B2)

Dissolve 10 mg of rat IgG in 10 ml of S2. Store in 0.5-ml aliquots at 4 °C for ≤1 year.

Blocking reagent 3 (B3) sheared salmon sperm DNA

This reagent comes at a 10-mg/ml concentration in ddH2O at −20 °C. Store in 0.5-ml aliquots at 4 °C for ≤1 year.

1× Tris-EDTA (TE) buffer

Add 1 ml of 1 M Tris (pH 8.0), 200 μl of 500 mM EDTA (pH 8.0) and 0.02% (wt/vol) NaN3 to 98.8 ml of ddH2O. Store at RT for ≤1 year.

BC4 solution

Prepare a mixture of the 57 nonmodified CODEX DNA oligonucleotides (Supplementary Table 1) by dissolving in TE buffer to a final concentration of 0.5 mM per oligonucleotide. Store in 0.5-ml aliquots at 4 °C for ≤1 year.

CODEX FFPE blocking solution

Add 50 μl of B1, 50 μl of B2, 50 μl of B3 and 70 μl of BC4 to 780 μl of S2. Mix gently by pipetting up and down. Store at 4 °C for ≤2 weeks.

CODEX staining buffer 4 (S4)

Add 50 ml of 5 M NaCl solution to 450 ml of S1. Gently mix. Store at 4 °C for ≤1 year.

Paraformaldehyde fixation solution Dilute 16% (wt/vol) paraformaldehyde (PFA) at a 1:10 ratio with S4 (vol/vol). Prepare fresh before each experiment and use a fresh vial of 16% PFA every 2 weeks.

BS3 aliquots

Dissolve 50 mg of BS3 in 250 μl of DMSO (ampule). Store in 20-μl aliquots at −20 °C for ≤6 months.

Final fixative solution Add 20 μl of BS3 (thawed to RT) to 1 ml of 1× DPBS. Mix gently by pipetting up and down. Prepare fresh before each experiment.

H2 buffer

Add 30 ml of 5 M NaCl solution, 10 ml of 1 M Tris (pH 7.5), 0.943 ml of Triton X-100, 2.03 g of MgCl2•6H2O and 0.02% (wt/vol) NaN3 to 960 ml of ddH2O. Store at RT for ≤1 year.

Hybridization buffer

Combine 100 ml of DMSO with 400 ml of H2 buffer. Stir gently to thoroughly combine. Prepare fresh before each experiment.

Stripping buffer

Add 62.5 ml of H2 buffer to 187.5 ml of DMSO. Stir gently to thoroughly combine. Prepare fresh before each experiment.

CODEX hybridization solution

For each tissue section, add 7 μl of B3, 1 μl of each complementary fluorescent oligonucleotide and the appropriate volume of H2 buffer to a final volume of 100 μl. Mix gently in an Eppendorf tube by pipetting up and down. Prepare fresh before each experiment.

Hoechst staining solution

Add 1 μl of Hoechst 33342 to 999 μl of H2 buffer. Mix gently in an Eppendorf tube by pipetting up and down. Prepare fresh before each experiment.

Plate buffer

Add 83.3 μl of Hoechst 3342 and 2.5 ml of B3 to 50 ml of H2 buffer. Store in the dark at 4 °C for ≤4 weeks.

Fluorescent oligonucleotide stock solution

Dissolve the lyophilized fluorescent oligonucleotide pellet in the appropriate volume of 1× TE buffer to a concentration of 100 μM (note: volume provided by IDT). Store in the dark at −20 °C for ≤2 years. Generally, we will order custom fluorescent reporter oligonucleotides from IDT that are HPLC purified, 1-μmol batch size and lyophilized.

Fluorescent oligonucleotide working solution

Dilute the fluorescent oligonucleotide stock solution at a 1:10 ratio with 1× TE buffer (vol/vol). Store in the dark at 4 °C for ≤1 year.

DRAQ5 staining solution

Add 2.5 μl of DRAQ5 to 247.5 μl of plate buffer. Prepare fresh when setting up the multicycle plate.