I'm trying to quantify focal adhesions in mouse embryo cryosections, E8.5. We have vinculin antibodies, I'm starting to test them. I can't find any published cross-sections of vinculin staining, I'm sure I won't simply be able to count individual focal adhesions.
I want to specifically look at focal adhesions in the surface ectoderm, but the treatment likely affects the whole embryo, so a backup could be western blotting, but I'm skeptical that would be sufficient.
I would try quantifying staining with paxillin as a reasonable proxy for FAs. phospho paxillin goes in the nucleus, but paxillin is never anywhere but in focal adhesions. look at my papers for what antibody we use. make sure you permeabilize and rinse the hell out of it, too. I would not use vinculin as suggested above, as it is in adherens junctions and tends to have a lot of background.
Dear Philip,
I don't think you can simply quantify FAs from cross-sections. Usually, you let the cells adhere to a sticky surface (e.g. fibronectin), where they can nicely form FAs that you then can visualize e.g. by TIRF microscopy. In the embryonic cross sections, the cells adhere to each other or to the surrounding matrix, but not in a single plane. Rather they are in positioned in 3-dimensions. So you can't visualize all connections, what you yould need to do in order for an accurate quantification.
Best regards,
Volker
Volker is correct. Typically focal adhesions are visualized in single cells are thin glass coverslips by interference reflection microscopy. We did not try to quantify focal adhesions per cell, but instead scored cells as positive or negative for focal adhesions. The best you can do is IHC for vinculin and the particular integrin that you think is involved. Unless the cell is attached to a basement membrane, I suspect that the primary interactions in an embryo are cell-cell, although some isoforms of laminin are expressed as early as the 4 cell stage.
I would try quantifying staining with paxillin as a reasonable proxy for FAs. phospho paxillin goes in the nucleus, but paxillin is never anywhere but in focal adhesions. look at my papers for what antibody we use. make sure you permeabilize and rinse the hell out of it, too. I would not use vinculin as suggested above, as it is in adherens junctions and tends to have a lot of background.
This can help:
Step-by-step quantitative analysis of focal adhesions
http://www.sciencedirect.com/science/article/pii/S2215016114200203
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models