Dear all,
I am having a problem with too strong mCherry fluorescent signal in vivo that can be seen by naked eyes. It bothers us because in immunostaining the red channel (594) interrupts with other channels so much (especially far red channel 647). Is there a easy way to get rid of the red fluorescent signal? I am thinking of using KMnO4 to treat the tissue.
Thanks a lot!
Zheng, would UV light do harm to other channel staining?
Fan, it is way too bright even without any staining for RFP.
Hi,
Possible alternative to KMnO4 is NaBH4. It will reduce fluorescent protein and remove the fluorophore. It is used in microscopy experiment to remove fluorescence.
Good luck,
Grazvydas
The best way to selectively bleach mCherry is to bleach near its excitation wavelength (~587 nm). Even with very different signal levels, you should be able to see far-red without significant mCherry background with the appropriate selection of excitation and emission filters/light sources. Specifically, exciting with light at longer wavelengths than ~620 nm should give very little mCherry excitation.
Also, I found that mCherry fluorescence is very poor in "Vectashield" mounting medium, while the stability of some small-molecule dyes is greatly enhanced (fixed E. coli samples). Perhaps that would solve your problem without requiring new filter sets or light sources. This is only an N=1 experiment, so caveat emptor!
Hi,
Grazvydas Lukinavicius what concentration do you use for NaBH4? how long do you expose/treat your samples?
also if I cannot obtain NaBH4, the same questions apply to KMnO4
thank you
Hi Yu-Chen, I wonder if you have the solution for this question? I am facing the same issue as you. Thanks!
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