Currently I am working on one protein with 2 different variants, it is basically nuclear localization, but there is no clue about the 2 variants. If I checked individually means either one may be in nuclear localization and another one is nuclear membrane. So what I did means, I used co-localization of protein (one protein + one variant) at a time using blue (DAPI), red (know protein), green (Variant). I got nice difference with the variant localization, but for publication i like to put all in one cell to present their localization. I am going to use confocal microscope, can anyone suggest me which way is better to work me in this matter...
Thank you for your valuable time and suggestions...
For 4 color staining, you could try adding in a secondary antibody with a fluorophore that emits in the far visible red. Examples would be Cy5, AlexaFluor633 or 647, and various others. These fluorophores have excitation/emission spectra that don't overlap too much with more visible, green-laser excited dyes such as rhodamine or Cy3. The only issue with using far visible red dyes is that they aren't terribly visible to the eye, so it is more difficult to determine if the staining worked just by looking through the eyepieces unless it is VERY intense. However, a camera or detector should still pick the signal up fine. The far red channel could then be pseudo-colored to something else like magenta or white.
Just another thought: If you're using triple antigen immunofluorescence, make sure your secondary antibodies have been screened for cross-reactivity to other species. You wouldn't want to get a false positive for co-localization by having an anti-mouse secondary partially binding to a rabbit primary.
For 4 color staining, you could try adding in a secondary antibody with a fluorophore that emits in the far visible red. Examples would be Cy5, AlexaFluor633 or 647, and various others. These fluorophores have excitation/emission spectra that don't overlap too much with more visible, green-laser excited dyes such as rhodamine or Cy3. The only issue with using far visible red dyes is that they aren't terribly visible to the eye, so it is more difficult to determine if the staining worked just by looking through the eyepieces unless it is VERY intense. However, a camera or detector should still pick the signal up fine. The far red channel could then be pseudo-colored to something else like magenta or white.
Just another thought: If you're using triple antigen immunofluorescence, make sure your secondary antibodies have been screened for cross-reactivity to other species. You wouldn't want to get a false positive for co-localization by having an anti-mouse secondary partially binding to a rabbit primary.
Hi Kevin,
Thanks for your valuable suggestion, very important point you mentioned secondary antibodies issue.
Thank you
I would suggest using alexa 488, 568 and 633 conjugated secondary antibodies, and in particular I recommend using donkey secondaries. DAPI displays the tendency to bleed into the 488, so sequential acqusition is the best option.
A good confocal can be pushed to at least 5 fluorochormes without resorting to spectral unmixing: 405, 488, 555, 594, 547 emission bands are easily achievable.
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