Immunofluorescence Protocol with Image-iT™ FX Signal Enhancer

A. Solutions and Reagents

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

• 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 8.0.
• Formaldehyde, 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh, store opened vials at 4°C in dark, dilute in PBS for use.
• Permeabilization Buffer (1X PBS/0.2% Triton X-100):
  To prepare 25 ml, add 2.5 ml 10X PBS, and 22.5 ml dH₂O and mix well. While stirring, add 50 µl Triton X-100.
• Image-iT™ FX Signal Enhancer (Invitrogen, Eugene, OR, cat# I36933)
• Blocking Buffer (1X PBS/5% normal goat serum/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.
• Antibody Dilution Buffer (1X PBS/1% BSA/0.3% Triton X-100):
  To prepare 40 ml, add 4 ml 10X PBS to 36 ml dH₂O, mix. Add 0.4 g BSA and mix well. While stirring, add 120 µl Triton X-100.
• Prolong^® Gold Antifade Reagent (Invitrogen, Eugene, OR, cat# P36930)

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed, and stained directly in multi-well plates, chamber slides, or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 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 (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Permeabilize the cells in Permeabilization Buffer for 5 minutes at room temperature.
2. Rinse three times in PBS for 5 minutes each.
3. Apply 3–4 drops of Image-iT™ FX Signal Enhancer (or sufficient volume to cover the cells) and incubate for 30 minutes at room temperature.
4. Rinse three times with PBS for 5 minutes each.
5. Block specimen in Blocking Buffer for 60 minutes.
6. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
7. Aspirate blocking solution, apply diluted primary antibody.
8. Incubate overnight at 4°C.
9. Rinse three times in PBS for 5 minutes each.
10. Coverslip slides with Prolong^® Gold Antifade Reagent.
11. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.