Fluorescent Western blots use secondary antibodies conjugated to different dyes with non-overlapping spectral emissions to simultaneously detect multiple proteins. These are the most important factors to consider when performing fluorescent Western blots: 

• Optimize the detection of each target separately before performing simultaneous detection when multiplexing. 
• Titrate primary and secondary antibodies to determine the best concentrations for each. Use a bot blot and checkerboard titration for this purpose.  
• Adjust antibody concentrations if necessary. You may need to increase primary antibodies to about 2-5 times higher than the concentrations used in chemiluminescent Westerns, while secondary antibodies may need to be adjusted to start at 1:5000 dilution. 
• Use low-autofluorescence membranes such as PVDF (Polyvinylidene Fluoride). Avoid using nitrocellulose membranes that can cause high background signals. 
• Use a pencil to mark the dot instead of inks or dyes such as Coomassie and bromophenol blue, which can autofluoresce.
• To prevent cross recognition in multiplexing, use primary antibodies from different species and cross-adsorbed secondary antibodies  
• Select fluorophores with distinct spectra to prevent overlap in multiplexing. 
• Skip a lane between fluorescent molecular weight markers and samples when loading, to prevent signal bleed from the molecular weight markers into the sample lanes. 
• Protect fluorescent antibodies by working with them on the bench but storing them in the dark. 
• For better signal clarity, detect the strongest target in the blue channel, weakest in the red channel, and medium in the green channel. 
• Store blots in the dark when archiving. 
• Cleanliness is vital to preventing background noise. Clean all trays and equipment thoroughly before use. Use powder-free gloves to handle the gel and membrane and keep trays covered during incubation.