I am trying to analyse the state of the gut epithelium by analysing by confocal microscopy the state of some adhesion proteins (to note, Claudin and occluding); however, these proteins are not exclusive of epithelial cells, which is why I would like to add a marker specific for this subset. Could you please recommend me any marker/combination of markers that makes easier the identification of epithelial cells? Thank you in advance
Hello,
You could use Epithelial Cell Adhesion Molecule (EpCAM).
In human intestinal epithelium, anti-EpCAM antibody stains the region of tight junctions (TJs) and the lateral membrane. The EpCAM signal can also be detected on the brush border.
For more details you could refer to the research article given below.
https://doi.org/10.3892/ijmm.2018.3764
I hope this helps.
Best.
Thank you so much Mr Nobre! I considered that as my first option because I have been using this marker to identify epithelial cells in Flow Cytometry. However, experiments trying to fine-tune this antibody to confocal microscopy have given me low-specificity staining. That is why I started to think about changing the antibody, but I will try much lower dilutions too see if I can obtain a good-quality staining. Thank you so much for your answer!
Hello Maria,
I agree with Malcolm, you should use anti-EpCAM antibody for staining. If you want to test another antibody which is related to tight junction, you can use tricellulin. Tricellulin is restricted to the tricellular contacts of enterocytes. You can also use E-Cadherin as an marker for adhesion of two enterocytes and is essential for morphogenesis of the intestinal epithelium....
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Article A Key Role for E-cadherin in Intestinal Homeostasis and Pane...
Best regards,
Constanze
Hi Maria,
I agree with other people that EPCAM and E-cadherin are good markers. The staining will be found at the lateral membranes of cells (E-cadherin has strong signal at adherens junctions and weak signal at lateral membrane). ZO-1 is also a good marker for tight junctions although it also stains blood vessels and mesothelium (You can distinguish them by morphology).
When you optimize your protocol, I would reccomend to try different fixation method. Most antibodies of tight junction proteins give great staining with ethanol (or methanol) fixation. Acetone treatment following ethanol fixation sometimes improves S/N ratio.
Good luck!