Dear Colleague,
I use PKH26 for cell tracking after hepatocyte transplantation. We found that if we look directly for red flurescence with DAPI staining, the labeled cells can be found much more than we take a look at them after co-staining with other antibodies. The signals seem lost after co-staining with other additional antibodies. Do we have any solution? Thanks in advance.
Hello ..
PKH26 is a fluorescent dye that can be used to label cells for tracking purposes. It is often used to label cells in cell transplantation studies, such as hepatocyte transplantation, to monitor the movement and behavior of the transplanted cells.
It is possible that the fluorescent signal from PKH26 is being lost after co-staining with other antibodies. This can occur if the additional antibodies bind to the same cell surface or intracellular epitopes as the PKH26, or if the additional antibodies interfere with the binding of the PKH26 to the cells.
There are a few strategies you can try to minimize this problem:
I hope this helps!
Mohammad Alzeyadi
Thank you, I used co-staining method 1 and 2. I am wondering whether the some steps in protocol of antibody staining will get PKH26 off. By the way, I did not see many co-stain photos in publication, maybe they encounter the same issue?
Hello
Co-staining with multiple antibodies can be challenging and can result in loss of fluorescence signal from one or more of the antibodies. The steps in the protocol of antibody staining can affect the fluorescence signal from PKH26.
For example, if the cells are washed or permeabilized with reagents that can also remove PKH26 from the cells or damage the dye, this will result in a loss of fluorescence signal. Similarly, if the cells are exposed to conditions that are not compatible with PKH26, such as high temperatures or high pH, this can also result in loss of signal.
It is also possible that other staining steps, such as treatment with secondary antibodies or counterstaining with other dyes, can interfere with the fluorescence signal from PKH26. As you pointed out, it is common that few co-stain photos are shown in the publication and it could be due to the loss of fluorescence signal or issues with the methods they used.
It is important to note that these are general considerations and the specifics of the protocol will depend on the particular cell type, PKH26 concentration and other factors and therefore it is best to test various protocols to identify the best one for your specific application.
Regards.
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