Are you mounting your sections undercover slip with a solution containing antifade compounds such as "Polymount" from poly sciences or the equivalent from other suppliers? We use Polymount and have very little fadi g problems with 486

Sorry, that's 586

Really now! That's 546

I use 555, not 546. No big change, but I recommend to use 555 if you use 488 or GFP simultaneously.

Anyway, in general, the selection of the dye depends on the excitation/emission of the microscope. Ask to the microscope manager or the dealer staff of the microscope company about about the filter set and the lamp information (excitation/emission, laser, mercury etc) and also recommended dye.

My two cents...Two factors to keep in mind: 1. Overall brightness is a factor of both QY and ext. coeff., so 546 will be brighter in this case and I would choose it unless 2. It may be more valuable for you to assess which will give you the best spectral separation from whatever else you may be looking at in the cells or which may be most appropriate to the microscope that you are using.

In our experience, A546 is more durable than A555 when used under similar conditions (same specimen type and mounting medium). In most cases, I would use A546 unless there was a possible "bleed through" problem that needed to be avoided. I suggest you use an anti-fade agent in your mounting medium (or buy one containing it). We use DABCO-glycerol or Vectashield if we require a more permanent mounting.

It is true that Alexa dyes do not benefit from anti-fade agents as much as other dyes, but this makes sense because they are designed to be less available to the oxidation reactions that bleach dyes - and which anti-fade agents protect against. The shift in excitation frequency toward the red in A555 may also increase its durability. In any case, we believe the operational difference between the two is small.

Thanks for the comments! I do use antifade mouting media (currently Dako Immunofluorescence Mounting Medium). Still, my Alexa546 stained slides recently started fading literally in a matter of seconds. I now think this must be because the antibody lot is too old. But anyway, I'm thinking whether I should just get a new lot of the Alexa546 secondary or perhaps switch to Alexa555. To clarify, I'm in the moment only interested in the signal intensity and photobleaching properties and not so much about other spectral properties.

I'm curious if you used AF555 or AF546 and your experience.