Photobleaching During Fixation and Storage

Symptom

Progressive loss of fluorescence signal over time, particularly noticeable in samples fixed and stored for later analysis. Light-sensitive fluorophores show dramatically reduced intensity.

1. 1 Samples exposed to ambient light during fixation and permeabilization steps
2. 2 Inadequate light protection during overnight storage of fixed cells
3. 3 Photobleaching of fluorophores during extended processing times
4. 4 Use of clear tubes instead of light-protected containers

1. 1 Keep samples protected from light during fixation and all subsequent steps
2. 2 Use aluminum foil or amber tubes to shield samples from light exposure
3. 3 Minimize time between staining and acquisition, even for fixed samples
4. 4 Store fixed cells at 4°C in darkness if overnight storage is required

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