Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

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

Stock Solutions

• 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.
• Ethanol, 70% solution, deionized.
• 1.5 M Hydrochloric acid.

• Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100):

  To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH₂O, mix well. While stirring, add 30 µl Triton™ X-100.

• Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 and 0.5 mL 20X PBS to 9.5 mL dH₂O. Mix well then add 0.1 g BSA (#9998), mix.

• 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
• Prolong^® Gold AntiFade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).

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

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

1. Aspirate media, cover cells completely with ice-cold 70% ethanol.
2. Allow cells to fix for 5 minutes at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
4. Add 1.5 M HCl and incubate for 30 minutes at room temperature.
5. Aspirate HCl and rinse two times in 1X PBS for 5 minutes each.
6. 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 for 60 minutes.
2. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
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.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
7. Rinse three times in 1X PBS for 5 minutes each.
8. Mount samples in an appropriate antifade reagent such as Prolong^® Gold Antifade Reagent (#9071) or Prolong^® Gold AntiFade Reagent with DAPI (#8961).
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.