Confocal microscopy and immunofluorescence protocols, optimization methods and techniques - is there an immunofluorescence or confocal microscopy protocol you want to share? Do you have some comments, protocols or suggestions regarding deconvolution or scientific imaging tips? We want your feedback.
Protocols: immunofluorescence, cryostat sections, sample preparation ...
Troubleshooting:weak signal, high background noise? Try some of these suggestions.
Weak Signal
• Increase antibody concentration or incubation time(this may increase nonspecific background signal)
• Observe the specimen using a conventional epifluorescence microscope: is fluorescence visible?
• Enlarge pinhole diameter (note: this will increase the optical slice thickness)
• Increase pixel dwell times by reducing scanning speed
• Increase excitation (laser) energy Note: the risk of bleaching and signal saturation are greater with greater laser power.
High Background Signal:
• Decrease antibody concentration or incubation time
• When preparing tissue sections (immunofluorescence protocol), perform stringent wash steps
• Use deconvolution software to limit the effects of light noise
• Use blocking steps during immunofluorescence (eg. BSA)
• Limit the laser power