PhosphoPlus^® In-Cell Duet (ICW Compatible) Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na₂HPO₄) and 2.4 g potassium phosphate, monobasic (KH₂PO₄) to 1 L dH₂O. Adjust pH to 7.4.
2. Formaldehyde, use fresh, dilute in PBS for use.
3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton X-100): To prepare 25 ml, add 2.5 ml 10X PBS, 1.25 ml normal goat serum and 21.25 ml dH₂O and mix well. While stirring, add 75 µl Triton X-100.
4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 25 ml, add 2.5 ml 10X PBS to 22.5 ml dH₂O, mix. Add 0.25 g BSA and mix well. While stirring, add 75 µl Triton X-100.

B. Specimen Preparation

NOTE: Cells should be grown, treated, fixed, and stained directly in multi-well plates.

1. Aspirate culture medium, and then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
   NOTE: Formaldehyde is toxic, use only in fume hood.
2. Allow cells to fix for 15 minutes at room temperature.
3. Aspirate fixative, rinse three times in PBS for 5 minutes each.
4. Proceed with immunostaining.

C. Immunostaining

NOTE: Include control well(s) for detection cocktail staining alone (no primary cocktail) for nonspecific background correction.

1. Block specimen in Blocking Buffer for 60 minutes.
2. While blocking, prepare primary cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary cocktail.
4. Incubate overnight at 4°C.
5. Rinse three times in PBS for 5 minutes each.
6. Prepare detection cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
7. Incubate 1–2 hours at room temperature in the dark.
8. Rinse three times in PBS for 5 minutes each.
9. For best results examine specimens immediately using appropriate excitation wavelengths.