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CY5.5/Alexa555 double

CY5.5/Alexa555 double - Immunofluorescence Forum

CY5.5/Alexa555 double - Immunofluorescence Forum. Talk, discuss and post questions on immunofluorescence techniques including paraffin, frozen sections or cultured cells.


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Old 02-25-2009, 11:38 PM
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Question CY5.5/Alexa555 double



Hi, I am new in IHC.
I was asked to triple stain microtubulin, GFAP and neurofilament in neuronal tissue with CY5.5/Alexa555 and 486. 486 showed up pretty good, the other two looked terrible. The two structural proteins are nearly overlapped (processes and neuronal fibers) or parallel lined up. Can the two fluorescence be separated with emission filters of
425 long pass
500+\- 20
535
610
Thanks
Sand
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Old 03-01-2010, 04:23 PM
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Default Re: CY5.5/Alexa555 double

It sounds like you're using a traditional fluorescent scope and not a confocal. When you say emission filters, do you mean filter cubes? A filter cube will tell you if the scope can detect or differentiate between the fluorescent signals. However, you also need to make sure that the light source for each filter set will excite the fluorfores being used.

If you look at this website*, you can select any combination of fluorfores and see exactly how their excitation and emission spectra overlap. You can then compare this to the filter set information on your scope.

*Actually, the forum will not let me post a website yet. Google search for "Invitrogen spectral viewer" ...it's the first link.

Also, BD bioscience has a good one.
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Old 03-01-2010, 05:27 PM
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Default Re: CY5.5/Alexa555 double

As major pointed out, you didn't provide enough info for us to tell you if you're scope will work. If using confocal, you need to tell us the laser wavelengths and emission filter values. If using widefield we need to know your excitation and emission filter pairs (i.e. cubes).

Generally speaking, the three colours you have picked can be imaged together quite well (assuming the proper filters & lasers). That said, Cy5.5 is not exactly a great flourophore; both alexa and dylight dyes of the same wavelength are available that are much better (brighter and less prone to photobleaching).

Assuming that your scope is set up to image these dyes, there are a few other things that could be happening:

1) Your antibodies may suck. This is more common than you may think - if possible try labelling a structure you know labels well (for example, whatever you're labelling with 486), but use a secondary that is 555 or Cy5.5 labelled. If you see good imaging you're issue is more with the primary antibody than with the label/scope. You can also do the reverse - work out the best labelling conditions, using a 486 secondary (and staining one antibody at a time).

2) You didn't specify what was "terrible" about your staining - faint, lots of background, not where it "should" be, etc. More detail in this area may help us address the question, but briefly:
  • Faint staining: try increasing antibody amounts (primary and/or secondary)
  • Bad background: try blocking longer, or use a different blocking agent
  • Protein localises "wrong": Antibody may cross-react with something else (try new primary), you may have discovered something cool (send manuscript to science)

3) You may want to check your cells, to make sure they are expressing your proteins of interest. I've known people who spend months trying to track down a staining issue, only to find that their cell did not express their protein-of-interest.

Hope this helps, and please provide us with the missing info about your scope.

Bryan
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