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.
- Permeabilization Buffer (1X PBS/0.2% Triton X-100):
To prepare 25 ml, add 2.5 ml 10X PBS, and 22.5 ml dH2O and mix well. While stirring, add 50 µl Triton X-100. - Image-iT™ FX Signal Enhancer (#11932)
- 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 goat serum and 21.25 ml dH2O and mix well. While stirring, add 75 µl Triton X-100. - Antibody Dilution Buffer (1X PBS/1% BSA/0.3% Triton X-100):
To prepare 40 ml, add 4 ml 10X PBS to 36 ml dH2O, mix. Add 0.4 g BSA and mix well. While stirring, add 120 µl Triton X-100. - Prolong® Gold AntiFade Reagent (#9071), 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.
- 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).
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.
- Permeabilize the cells in Permeabilization Buffer for 5 minutes at room temperature.
- Rinse three times in PBS for 5 minutes each.
- Apply 3–4 drops of Image-iT™ FX Signal Enhancer (#11932) and incubate for 30 minutes at room temperature.
- Rinse three times with PBS for 5 minutes each.
- Block specimen in Blocking Buffer for 60 minutes.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in PBS for 5 minutes each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071), with DAPI (#8961).
- For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.