Before you start
- Seed cells on coverslips (coated if needed) 1–2 days before.
Reagents and materials
Totals are for 6 coverslips (+10% extra). Per-coverslip amounts are in the middle column.
| Reagent | Per coverslip | Total |
|---|---|---|
| 4% paraformaldehyde in PBS | 0.5 mL | 0.5 mL |
| PBS (washes) | 6 mL | 6 mL |
| Permeabilisation buffer (0.1–0.3% Triton X-100 in PBS) | 0.5 mL | 0.5 mL |
| Blocking buffer (5% normal serum or 3% BSA in PBS) | 0.5 mL | 0.5 mL |
| Primary antibody in blocking buffer | 100 µL | 100 µL |
| Fluorescent secondary antibody (1:500–1:1000) | 100 µL | 100 µL |
| DAPI (1 µg/mL) | 0.3 mL | 0.3 mL |
| Antifade mounting medium | 10 µL | 10 µL |
Buffers and recipes
PBS (1×) per 1 L
| NaCl (137 mM) | 8.0 g |
| KCl (2.7 mM) | 0.2 g |
| Na₂HPO₄ (10 mM) | 1.44 g |
| KH₂PO₄ (1.8 mM) | 0.24 g |
| Ultrapure water | to 1 L |
Adjust to pH 7.4 and autoclave. This PBS is Ca²⁺/Mg²⁺-free (use it for detaching cells).
Equipment
- Glass coverslips in a multiwell plate
- Humid chamber
- Fluorescence or confocal microscope
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Wash
Rinse gently with PBS.
Fix
Fix with 4% PFA for 10–15 min at room temperature (or ice-cold methanol for 10 min at −20 °C for some antigens).
Wash
Wash 3 × 5 min with PBS.
Permeabilise
Incubate in 0.1–0.3% Triton X-100 for 10 min (skip after methanol fixation).
Block
Block for 1 h at room temperature.
Primary antibody
Incubate with primary antibody overnight at 4 °C in a humid chamber.
Wash
Wash 3 × 5 min with PBS.
Secondary antibody
Incubate with secondary antibody for 1 h at room temperature in the dark.
Counterstain
Wash, then DAPI for 5 min, and wash again.
Mount and image
Mount on slides with antifade medium, let cure and image with identical settings for all conditions.
Critical points
- Controls: secondary-only, and a known positive and negative cell type.
- Use serum from the secondary antibody’s host species for blocking.
Troubleshooting
| Problem | What to check or change |
|---|---|
| High background | Block longer, titrate antibodies, wash more, check for autofluorescence. |
| No signal | Try another fixation method (methanol vs PFA), check antibody validation, increase concentration. |
Safety
PFA is toxic and a sensitiser: use a fume hood or ventilated area.
Further reading
- Donaldson JG. Immunofluorescence staining. Current Protocols in Cell Biology (2015).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.