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.
Fixing/Blocking
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
Solutions
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.
