Immunofluorescence staining
Fix cells, permeabilise membranes and detect the target with fluorescent antibodies. The right fixation and permeabilisation depend on the target, so compare two methods side by side at first.
Key points
- Fixation and permeabilisation must suit the antigen
- A secondary-only control reveals background
- Protect from light and image every sample with identical settings
Materials
- Cells on coverslips or chamber slides
- PBS
- Fixative: 4% PFA or cold methanol
- Permeabilisation: 0.1–0.3% Triton X-100 in PBS
- Blocking: 5% normal serum or 3% BSA
- Primary antibody, fluorescent secondary
- Nuclear stain (DAPI or Hoechst)
- Anti-fade mounting medium
- Fluorescence microscope
Steps
🔬 Variable explorer — what changes if…?
The skeleton stays the same, but concentrations, times and reagents change between experiments. Click an item to see what it affects and compare two conditions (A and B) side by side.
Fixation
What it isHow proteins are locked in place.
ShorterToo short: structure collapses and antigen washes out.
LongerPFA too long: epitopes are masked and signal weakens.
Typical choiceFollow the antibody datasheet; otherwise compare PFA and methanol side by side.
Permeabilisation (Triton)
What it isOpens membranes so antibodies can enter.
MilderToo mild: antibodies cannot enter and intracellular signal is weak.
HarsherToo harsh: membranes are destroyed and membrane proteins lost.
Typical choice0.1–0.3% for 5–10 min. For surface proteins use a mild detergent (saponin) or skip it.
Primary antibody dilution
What it isSignal and background both follow antibody concentration.
More concentratedToo concentrated: the whole cell glows and localisation is lost.
More diluteToo dilute: weak signal needs long exposure, adding noise.
Typical choiceStart at the IF dilution on the datasheet (often 1:100–1:500) and compare 2–3.
Blocking
What it isCovers sticky sites so antibodies bind only the target.
Too littleToo little: signal even in the secondary-only control.
Typical choice5% normal serum from the secondary host species (e.g. goat secondary → goat serum), 30–60 min.
Light exposure · imaging
What it isFluorophores fade with light exposure (photobleaching).
More lightLong illumination or varying exposure: brightness comparisons become invalid.
Typical choiceKeep dark, use anti-fade, identical exposure and gain, no saturated pixels.
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| No signal | Fixation/permeabilisation unsuitable, antibody too dilute | Try another fixation, run a positive control, more antibody |
| High background | Poor blocking, non-specific secondary | Secondary-only control, dilute more, wash more |
| Signal bleeds into another channel | Spectral overlap | Image channels sequentially, check filters, pick separated fluorophores |
| Signal fades quickly | Photobleaching | Anti-fade medium, minimal light, image quickly |
⚠ Safety PFA is toxic and a suspected carcinogen — handle in a fume hood. Methanol is also toxic.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
FISH & fluorescence image counting → Group comparison statistics →