Immunofluorescence Protocol for Labeling with BrdU Antibody

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.
• BrdU (5-Bromo-2′-deoxyuridine) EMD biosciences (Cat. #203806)
• Ethanol, denatured 70%, Solution, American Bioanalytical (Cat. #CU00844)
• 1.5 M Hydrochloric acid
• 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 serum from the same species as the secondary antibody (e.g., normal goat serum, normal donkey serum) and 21.25 ml dH₂O and mix well. While stirring, add 75 µl Triton X-100.
• Antibody Dilution Buffer (1X PBS / 1% BSA):
  To prepare 40 ml, add 4 ml 10X PBS to 36 ml dH₂O, mix. Add 0.4 g BSA and mix well.
• 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.

BrdU Incorporation: Dilute BrdU in fresh, pre-warmed growth medium to a final concentration of 0.03 mg/mL. Add mixture to cells and incubate at 37°C for 30 minutes.

1. Aspirate media, cover cells completely with cold 70% ethanol.
2. Allow cells to fix for 5 minutes at room temperature.
3. Aspirate fixative, rinse three times in PBS for 5 minutes each.
4. Add 1.5M HCl and incubate for 30 minutes at room temperature.
5. Aspirate HCl and rinse two times in 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 datasheet in Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 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 in PBS as in step 5.
8. Coverslip slides with Prolong^® Gold Antifade Reagent or apply just enough to cover cells in multiwell plate.
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-Mouse

• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4409
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410