We wanted to study the cell bio-distribution in our animal models and we have used PKH26 in order to tag our cells. The cells can be observed in vitro using a fluorescent microscope (using attached cells on culture flasks), however things were not the same in vivo. It seems the cells could not be detected. We are sure that the bio-imaging system has the filter to view according to wavelength of PKH26, and yet we are now not sure what went wrong. Could the dye concentration be the reason? Requesting for your kind advice.
Thanking you all in advance.
Regards,
Naresh.
Hi,
I have used PKH26 to tag the cells in vitro and transplanted the same to rat model. we checked the migration of the cells by doing tissue slicing as described in immunohistochemistry. we could find the stained cells in liver, lungs and muscles hence i think the signal of PKH26 is strong enough in vivo even after weeks and its proportional to the thickness of the tissue. therefore detecting signal through imaging might take few standardizations.
Hello Nareshwaran,
Agree with Kavina as PKH26 is stable in vivo for quiet longer durations and it can retain 10% of its original fluorescence even after 6 months thats why its a reliable dye for long term in vivo tracking experiments. To rule out the possibility of problem in imaging system, you should use a positive control as many fluorochromes gets excited at that peticular wavelength. Once you get assured of system working properly, you can use a temporal imaging starting from 0 day to day of experiment deadline. If u r not getting fluorescence at zero day then the problem is with PKH26. It once happened in our system and using a new aliquote of PKH26 solved the problem very well because sometimes there may be degradation over time. So try to use a new aliquote in that case.. Hope it helps..!
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