Have been using Wheat Germ Agglutinin on cardiomyocytes to stain the plasma membrane, while also permeabilizing afterwards to stain intracellular targets.(Triton-X 0.1%)
Have been consistently getting endosomal,perinuclear and sometimes nucleus staining with the WGA(2ug/mL in Hanks/PBS), even after fixing before and after the WGA staining with PFA 4%(10 minutes , @RT, no methanol).
Have tried increasing the number of washes after WGA staining already, and also tried adding Normal Goat Serum during WGA staining(5%). Have tried doing the WGA staining with cold Hanks, and the washes before and after with cold Hanks to decrease any vesicle trafficking when the cells were live.
An example of the perinuclear and nuclear staining attached.
Thanks in advance for any suggestions!
Hi Joseph, I think what you are seeing is a process called "capping". It has been observed with Abs against cell surface proteins. Con A also induces capping because it is multivalent, like WGA.
Multivalent agents that bind cell surface proteins are aggregated. These aggregated surface proteins are internalized and deposited into endosomes and then lysosomes. These vesicles can appear to be everywhere in the cells.
Endocytosis is pretty quick at RT and I don't think that cold washes will help. Adding N-acetyl-D-glucosamine, which is a competitor for WGA might help, but you might want to think about staining cell membrane proteins directly with FITC or other amine reactive dyes.
I don't know why this happens but I'm curios to know about this "capping" process in general. If the cells are fixed prior (4% PFA) and also after, how can there be internalisation? It is not an active process?
Could it be that the first PFA fixation step you are doing also permeabilises the cells (for example some PFA if not of high quality, will also have methanol) and thus you are labelling - as expected - also endomembrane compartments? Maybe you could have a sample and try to image the sample directly after the labeling (PFA - label WGA - wash - image) to see if the problem is already at this stage.
Dear Joseph,
in my experience, 2 microgram/mL is a far to high concentration for plasma membrane staining with WGA. Try a tenth of this concentration. After fixation, you should not observe any capping of your cells. Why do you add normal goat serum? I can not see, why it should have any effect.
Regards, Fred
Thank you for your answers. We have improved the WGA staining by doing live cell staining for a very short period of incubation(1 minute), and also by post-fixing the WGA, and by decreasing the exposure to our permeabilization reagent(Tween20 or Triton) from overnight with the primary antibody to only 5 minutes before the primary antibody.
The quality of the monolayer seems to be extremely important as well. Thanks again!
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