Dear colleagues,
I am trying to take images of Vero cells with confocal microscopy. I have a memrbane dye (WGA, a lectin) that I use. However, in most of the cases the cells look like the plasma membrane has gone and only vesicles and the nuclear membrane are stained (the attached images). The cells are stained with 1 µg/ml WGA-A647 for 10 sec and then fixed with 4% PFA in PBS for 10 min. Afterwards I do an antibody staining at 37° C for 1-2 hrs. Could that cause this problem?
Thanks for any help.
WGA will be internalized as the membrane is internalized while the cells are alive, which explains the vesicles that you are seeing. It is not essential to stain with WGA while the cells are alive. Do you only want to see the membrane? (or at least majority only plasma membrane)
If so, you should fix first, then stain with WGA.
Also note that since you are doing an antibody stain you are probably permeabilizing. Strong detergents such as Triton X-100, especially at concentrations >/= 1%, will extract lipids and lipid rafts from the membrane, pulling WGA-stained sections off along with it. This gets worth with longer time, greater concentrations and higher temperatures. You may have to optimize these, or go with weak detergents such as Tween-20, or other permeabilizing agents like saponin.
Dear Jordan,
thanks for your response. Of course I started out with fixing my cells first but I had the feeling that fixation is part of the problem and I got better results staining first. And since I stain for only 10 seconds I doubt that active internalization plays a big role. I permeabilize the cells with 0.25 % saponin. But I have the feeling that none of that is responsible for these weird results as I sometimes see cells labeled exactly how I want it (nice staining of the plasma membrane only) but I cnannot reproduce it. No matter what I try...
Any other thoughts on that??
Thanks
Hi Tobias,
if your goal is to stain the membrane, maybe it would be better to try another stain. I've also had some problems with WGA and switched to Cholera Toxin B conjugates - it's very simple and works on most types of cells both alive and fixed. (ChTB binds to ganglioside GM1 localised in membrane - I don't know Vero cells but for example non-differentiated HT29 are not stained well. But it's the only cell line I can think of now.)
Hi, Tobias, I developed the protocol for this usage of WGA when I was in R&D here at Molecular Probes (I'm now in Tech Support). Manual: http://tools.thermofisher.com/content/sfs/manuals/mp00831.pdf
What you are seeing is certainly strange. First off, I wouldn't expect that only 10 sec would be long enough to get suitable labeling. For live cell labeling, it's usually on the timeframe of 10 minutes. Yes, it can traffic into the cells with endocytosis, leading to vesicular labeling, but 10 sec certainly isn't enough time to see the pattern you do. Fixing first would be the way to go, as Jordan suggested, but I see your comment that you tried this.
One thing to consider: you will want to use methanol-free formaldehyde ("formalin" for instance is 37% methanol-stabilized). That little bit of methanol permeabilizes the sample, leading to intercalation of the dye. Also, make sure the detergent is not in the fixative (some people combine them instead of separate steps).
Make sure to do a single-color control, to rule out the possibility that what you are seeing is actually bleedthrough from the dye used to detect your antibody.
If all else fails, you can consider using Concanavalin A conjugates instead, though the protocol would be comparable to what I would recommend for WGA. However, ConA isn't quite as uniform in distribution and label pattern on the plasma membrane.
My opinion respectfully differs from Martin in that CTB is more of a lipid raft label, which is going to be even more domain-like (and thus less uniform) on the plasma membrane.
Hi,
one easy dye for membrane staining is DIOC (3,3'Dihexyloxacarbocyaninjodid; we order it from Sigma):
stock DiOC = 0.5 mg/ml in ethanol
staining solution = 0.5 micrograms/ml - (in media or PBS)
stain cells for 10 min
rinse in media or PBS
observe in fluorescence with the fluorescein filter set
Problem: This stain will also stain inner membranes (especially ER). But from our experience the plasma membrane will be nicely stained as well.
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