I need to detect tight junction proteins in HUVEC. I heard tight junctions take time to form, and that I should be waiting for a few days to be able to detect the junctions. How long is that, particularly for ZO-1 expression?
Maria, you should not need to wait days to see ZO1 expression. Perform WB or qPCR at any stage in their growth and you will probably see some ZO1. I should point out though that you are likely to observe a higher abundance of ZO1 mRNA and protein when the cells are confluent (I have observed a similar phenomenon with E-cadherin). If you want to perform ICC for ZO1 and want to see strong lateral cell surface staining, then your best bet is to allow the cells to grow to confluence and stain on the same day they become confluent. However, waiting a day or two afterwards can't hurt. If you do want to perform ICC I recommend using solutions containing divalent cations as these will aid in the retention of cell-cell contacts - you'll get nicer results that way.
Best of luck.
Thank you for your answer! I found it very complete and useful
Hi Maria,
I start to see a nice ZO-1 expression after 2/3 days they have reached confluency. You can also wait more and keep seeing its expression over time. I usually seed 50K cells/cm2, I reach confluency the 2nd day and after that I start seing tight junctions. Hope it helps! Please let me know if it works also for you.
Best of luck!
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