I have a Leica SP2 microscope, so i can choose without problems both emission ranges. I would like to minimize the overlap between probes.
I would avoid FITC in the first place as it photobleaches pretty quickly, consider Alexa488. After that TRITC is pretty stable and quite bright as is the Alexa dye. I've not used the qdot 665.
Alexa 555 works very well (spectral mode with AOBS), but Alexa 633 could be used to avoid crosstalk totally
Hi Giuseppe,
Practically all of the choices that you listed will work. Most of the time I use FITC or Alexa488 and for the second color TRITC, TexasRed or Alexa594. It works great. As for Alexa633 and 655, these are in the far red range of the spectrum. We have Olympus confocal and because of the lack of the ultraviolet laser we are forced to use far red Alexa 647 for triple color staining. I dont know if your Leica SP2 has filters to separate the far red channel. I suspect that it does so all of the red fluorescent dyes that you listed should work.
Also, with any combination you have to use antifade. We are using ProLong Gold from Invitrogen.
I hope that I was helpful... Good luck!
Robert
I have only used Alexa 488 and 594, but in my experience they are far more photostable than FITC or Texas Red. Although I have only ever used them with an antifade reagent.
Since the Leica SP2 has spectral capabilities I would recommend the Alexa 633, Texas Red tends to be somewhat less photostable and can exhibit spectral shifts in different microenvironments. Qdots would also likely work provided that your application is not hindered by the bulky Qdot. (also note that you will excite the Qdot with whatever green light you excite your other fluorophore with so overlap might be a problem even with the spectral imaging system). Like the others, I would avoid FITC. It will limit you in terms of stability. Go for Alexa or Atto dyes. Even Cy3 should work in that range.
I agree with all comments regarding avoiding spectral overlap and using more photostable dyes assuming you are using microscopy. Although Texas Red might work, it will definitely photobleach much quicker than FITC based on my personal experience...
do you have to use FITC for your exp? FITC is easily bleaching and comparable intensities will be a pre-requisite for proper co-localization analysis- otherwise your analysis will be biased for one of the dyes. if you could use alexa488 it would be better. combine it with mCherry or TexasRed. YOu will be most safe for bleaching artefacts with Cy5 or Alexa633.
josef
It clearly depends on the power of the confocal lasers.
In a standard Carl Zeiss LSM 700/710 I would go for FITC/Alexo 488 and Alexa 633.
Is a way to prevent cross bleaching (the detectors are not 100% efficient).
Use sequential scan.
We have good success with our own NorthernLights dyes: NL557 (this one is very bright and stable), NL493 (replacement for FITC) and NL637 (far red). Since there are no "unbleachable" dyes we always use mounting media (NorthernLights Guard Mounting Media, Cat #NL996). You can read more about these reagents at R&D Systems web site (www.rndsystems.com).
Please give up on FITC!!!! Alexa488 is much more stable. For the second antibody all your choices seem ok. Just go to the spectra viewer on invitrogen website and play with the different combination.
Alexa 488 with either Alexa 594 or 633 are good choices provided you have the right laser lines and good bandpass filters
Giuseppe,
If you are set on using a green channel for your primary antibody, then I would advice Alexa 488. One of my favorite secondary antibodies for red channel is Alexa Fluor 568. Ample washing of samples in between antibody incubation and filtration of antibody/dilution buffer is highly recommended! Best of luck.
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