Fluorochrome Alexa Fluor

If you do an IF staining against XFPs, make sure the secondary antibody carries a fluorochrome that matches the XFP fluorescence (e.g., Alexa-Fluor 488 and GFP) to intensify the autofluorescence and to avoid cross talk with other secondary antibodies in double staining. 

Fluorochrome-conjugated secondary antibodies Alexa Fluor 488 (green)-, Alexa Fluor 594 (red)-, Cy2 (green)-, Cy3 (yellow-green)-, Cy5 (red)-conjugated species-specific antibodies (e.g., Jackson ImmunoResearch Laboratories, Newmarket, UK, or Molecular Probes , Life Technologies ). 

Immunofluorescence species-specific secondary antibodies (1 500) conjugated with fluorophores at different excitation wavelengths depending on the microscope (e.g., 488 nm for green or 594 nm for red fluorescence). Fluorochrome-labeled secondary antibodies can be obtained from different suppliers, e.g., Alexa Fluor, Invitrogen or DyLight Fluor, Thermo Fisher Scientific, Waltham, MA (caution light sensitive, store at4°C). 

Fluorochrome-conjugated secondary antibodies can be selected according to the specific needs of the study. The use of the so-called third-generation fluorochromes, e.g., Alexa Fluor , can also be used for improved efficiency. 

A) List of primary antibodies used for the figures. (B) Example of a combination of Alexa-Fluor fluorochromes that have nonoverlapping excitation and emission wavelengths... 

---