Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix. Adjust pH to 8.0.
2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
3. Saponin, reconstitute in dH₂O for use.
4. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.1% Saponin):
   To prepare 10 ml, add 0.5 ml 20X PBS, 0.5 ml normal serum from the same species as the secondary antibody (e.g., normal goat serum, normal donkey serum) and 9 ml dH₂O and mix well. While stirring, add Saponin to final concentration of 0.1%.
5. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.1% Saponin):
   Add 0.1 g BSA to 10 ml 1X PBS and mix well. While stirring, add Saponin to final concentration of 0.1%.

6. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

   • 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^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414
7. Prolong^® Gold AntiFade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).

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 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 1X 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 Blocking Buffer (containing 0.1% Saponin) for 60 minutes.
2. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer (containing 0.1% Saponin).
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 minutes each.
   NOTE: If using primary antibodies directly conjugated with Alexa Fluor^® fluorochromes, skip to step C9.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer (containing 0.1% Saponin) for 1–2 hours at room temperature in dark.
7. Rinse in 1X PBS as in step 7.
8. Coverslip slides with Prolong^® Gold Antifade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).
9. For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.