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.
- 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.
- Allow cells to fix for 15 minutes at room temperature.
- Aspirate fixative, rinse three times in PBS for 5 minutes each.
- Proceed with Immunostaining (Section C).
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.
- Block specimen in Incubation Buffer for 60 minutes.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Incubation Buffer.
- Aspirate Incubation solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in PBS for 5 minutes each.
NOTE: If using primary antibodies directly conjugated with Alexa Fluor® fluorochromes, skip to step C8.
- Incubate specimen in fluorochrome-conjugated secondary antibody* diluted in Incubation Buffer for 1–2 hours at room temperature in dark.
- Rinse in PBS as in step 5.
- Coverslip slides with Prolong® Gold Antifade Reagent.
- 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 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
posted January 2009
revised December 2010