Immunofluorescence Protocol

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.
• Incubation Buffer (1X PBS / 5% BSA / 0.3% Triton X-100)
  To prepare 25 mL, add 2.5 mL 10X PBS to 22.5 mL dH₂O, mix. Add 1.25 g BSA and mix well. While stirring, add 75 μL Triton X-100 (100%).
• Fluorochrome-conjugated secondary antibody (recommended secondary antibodies)
  NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
• 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. Block specimen in Incubation Buffer for 60 minutes.
2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Incubation Buffer.
3. Aspirate Incubation solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in PBS for 5 minutes each.
   NOTE: If using primary antibodies directly conjugated with Alexa Fluor^® fluorochromes, skip to step C8.
6. Incubate specimen in fluorochrome-conjugated secondary antibody* diluted in Incubation Buffer for 1–2 hours at room temperature in dark.
7. Rinse in PBS as in step 5.
8. Coverslip slides with Prolong^® Gold Antifade Reagent.
9. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.

*Recommended Secondary Antibodies:

Anti-Rabbit

• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4413
• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414