Infrared secondary? or Heellllpppp meeeee!

I am currently in 3-color hell.  I need an anti-mouse secondary that I can use alongside GFP and RFP.  I’ve tried Alexa350 (blue) and Alexa680 (infrared).  Neither worked for shit.  Anybody out there have a suggestion?

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9 thoughts on “Infrared secondary? or Heellllpppp meeeee!

  1. Someone from our lab likes a molecular probes Marina blue secondary. I tried it once and it didn’t work super well, but it was dimly okay. I think they sell it anti-mouse. Might be worth a shot if you’re desparate. (I used it with a DAPI epi-fluorescence cube, the person from my lab looked at his on our confocal, but I don’t know the specs on that visualization. It’s new and since I’m leaving soon, I haven’ t bothered to learn anything about it.

    good luck!

  2. The Alexa350 was very, very dim. I had to do a 7 second exposure just to see the damn thing. There was no way I could take a Z-stack with it because it bleached about half-way through. This was on a regular Zeiss fluorescence scope. The confocal doesn’t have a laser to deal with the blue fluorophore or I might have tried that since bleaching wouldn’t be as much of a problem.

    The Alexa680 didn’t give me any signal at all. Or rather, the background was higher than the actual specific signal. This was on the confocal (the other scope doesn’t have an IR filter set). I might try working with it a bit more just in case something went horribly wrong with my experiment.

    At the moment, it’s looking like I might be doing more cloning and swapping GFP and RFP with CFP and YFP and then I can use the Texas Red-X secondary which I’ve had great success with. But that will mean it will be weeks before I can have the data to put in the paper.

  3. I’ve never been able to get the Alexa 350 to work, MP once suggested to me that we use a 1:50 dilution for it.

    Is your RFP the standard, or are you using something like DsRed, or HC red?

    You might be able to get away with Alexa 647, which I use all the time. We usually use it with Alexa 594 (which is closer to the DsRed variants).

  4. Geeka: I tried 1:50 for Alexa 350 and I got cells that were blue all over. Not exactly what I was going for.

    As for the RFP, it’s DsRed monomer. Probably Alexa 647 would work. Where do you get it from?

  5. We’ve had good luck with Alexa 405. If it’s too dim, then your filter cubes aren’t optimized, so ask around to see if someone else has the filter set you need….I think we mostly use 1:200 for immunos.

  6. What about some of the Cy dyes (from Jackson I think)? Like Cy5? Hope you get it to work

  7. I’ve used Cy5, too, or Alexa594 or 633. Good luck!

    (Hi. I got here via a link from someone who posted to the scientiae carnival.)

  8. Cy5 and Alexa 647 should be good.

    But it sounds to me like your problem is FILTERS or POLARIZERS, not dyes.

    You’re not going to see every dye on every system, no matter what the sales person told you.

    Check your wavelengths carefully.

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