Table 1 Recipes for IF staining, antibody conjugation, and PhenoCycler staining solutions

10X Tris-EDTA Antigen Retrieval Buffer, pH 9.0

6.05 g Tris

1.85 g EDTA

400 mL ddH₂O

 - Adjust to pH 9.0

 - Complete to 500 mL with ddH₂O

Store at 4 °C for up to 30 Days

1X Tris-EDTA Antigen Retrieval Buffer, pH 9.0

50 mL 10X Tris/EDTA Buffer pH 9.0

450 mL ddH₂O

250 µL Tween20

 - Mix well and make fresh.

10X Tris Buffered Saline (TBS)

80 g NaCl

2 g KCl

30 g Tris

 - Adjust pH to 7.4

 - Complete to 1000 mL with ddH₂0

IF Wash Buffer

200 mL 10X TBS

800 mL ddH₂O

250 uL Tween20

Primary Blocking Buffer

1000 µL IF Wash Buffer

20 µL Goat or Donkey Serum

 - Vortex to mix.

FC Blocking Buffer

500 µL FC Block

5 µL Anti-Mouse HRP

 - Vortex to mix.

Antibody Buffer

1000 µL IF Wash Buffer

1 µL Goat or Donkey Serum

 - Vortex to mix.

Prepared DAPI

500 µL PBS

1 µL 1 mg/mL DAPI

 - Vortex to mix.

Antibody Reduction Master Mix (A)

(for 1 conjugation)

6.6 µL Reduction Solution 1 (A)

275 µL Reduction Solution 2 (A)

 - Thawed aliquots of Reduction Solution 1 (A) should not be re-used

Bleaching Solution

0.8 mL 10 M NaOH

2.7mL 50% H₂O₂

26.5 mL 1X PBS

Staining Buffer with Blockers (A)

(for 2 samples)

362 µL Staining Buffer

9.5 µL N Blocker (A)

9.5 µL G2 Blocker (A)

9.5 µL J Blocker (A)

9.5 µL S Blocker (A)

Post-Staining Fixation (A)

1 mL 16% PFA

9 mL Storage Buffer (A)

Final Fixative Solution (A)

1000 µL 1X PBS

20 µL Fixative Reagent (A)

 - Thawed aliquots of Fixative Reagent (A) should not be re-used

Screening Buffer (A)

3.5 mL 10X PhenoCycler Buffer (A)

24.5 mL Nuclease-Free Water

7 mL DMSO

 - Allow the Screening Buffer to equilibrate to room temperature prior to use

Reporter Stock Solution (A)

(for 5 cycles)

1220 uL Nuclease Free Water

150 uL 10X PhenoCycler Buffer (A)

125 uL Assay Reagent (A)

5 uL Nuclear Stain (A)