Immunofluorescence of Cultured Cells (Not Tissue)
This is our basic starting protocol for immunocytochemistry. Antibody dilutions, blocking and permeabilization times, and antibody incubation times can be empirically determined.
1. Wash cells 3-4x (5 min each) with PBS
2. Fix with 4% formaldehyde at room temperature for 20 min
3. Wash 3-4x (5 min) with PBS. At this point, fixed cells may be stored in PBS at 4 degrees C for several weeks or more
4. (optional depending on antibody) Permeabilize 0.05-0.3% Triton X-100 (or NP-40 or Tween-20) for 30 - 60 min in blocking buffer (recipe below). For B-TubIV (cilia marker), permeabilize w/ 0.3 % Triton.
5. Incubate with blocking buffer [no Triton] at 4°C for 1 hr (up to overnight). This extra blocking step is not always necessary
6. Wash 3x (5 min) with PBS
Antibody Incubation Steps
1. Incubate with primary antibody (diluted in blocking buffer) at 4°C for 4 hrs up to overnight
2. Wash 3-4x (5 min) with PBS
3. Incubate with fluorescently labeled secondary antibody (diluted 1:500 or 1:1000 in blocking buffer) at 4 °C for ~2 hrs); the lasers we have at the VA are 405 (for DAPI/Hoescht), 488 (AF488), 559 (for AF555), 635 (for AF547). Filters on the spinning disk in our lab work for AF488 (FITC), AF546/555 (TRITC), AF647 (Cy5) for triple labeling. You could also label on the DAPI channel for quadruple labeling with AF350 or 405.
4. Wash 3-4x (5 min) with PBS
5. DAPI/Hoescht stain if desired. We generally use mounting media with DAPI (Fluoroshield ab104139), which precludes this step.
6. Mount on slides using mounting medium
Blocking/Antibody Incubation Solution
1X PBS + Ca2+/Mg2+ pH 7.4
+ 0.2% saponin (for mild permeabilization; keep in for the duration of staining)
+ 2-5% Normal Serium (donkey or goat; use the secondary antibody species)
+ 1% BSA
4% Formaldehyde Solution
• 1. Dissolve 4 g of paraformaldehyde in 90 mL of H2O in a 50-mL beaker (hood).
• 2. Heat at 60°C (approximately) until dissolved on hot plate with stirring. Add a few drops of 1 M NaOH to help dissolve once you hit 55-ish °C
• 3. Cool to room temperature
• 4. Add 10 mls 10X PBS (no Ca2+)
• 5. Filter with a 0.22-μm disposable syringe filter
Instead of making this from powder, we routinely buy 32% formaldehyde solution (Electron Microscopy Sciences) and aliquot, freeze at -20°C, and make fresh 4% formaldehyde from that for weekly use.