Planning your experiments
Controls, controls and more controls!
Appropriate controls are critical for the accurate interpretation of immuno-staining results. See these pages R&D Systems and Sigma-Aldrich websites. This publication also describes controls for immuno-staining in details: Controls for immunocytochemistry: an update. Burry RW. J Histochem Cytochem. 2011 Jan;59(1):6-12. Review.
Some essential reading:
Spectral imaging and linear unmixing permit the separation of strongly overlapping emission spectra (i.e GFP and YFP) as well as the removal of autofluorescence (natural autofluorescence or fluorescence induced by the use of fixatives or DNA transfection reagents). For an introduction on the principle of spectral imaging and linear unmixing, go to the Zeiss Campus website.
Preferentially, emission spectra of mono-labelled samples are collected and used as reference spectra to unmix the multi-labelled sample. If no mono-labelled samples are available, the references spectra may be obtained manually on the multi-labelled image or automatically extracted by the software, although it can lead to wrong results.