I have immortalized Human Brain Endothelial cells. I am trying to stain VE-Cadherin and Occludin in this PFA fixed cells. Instead of getting staining at cell periphery (On cell membrane), I am getting staining in the cytoplasm. I had tried different fixatives like PFA and methanol, with and without permeabilization, permeabilization with different concentration of triton x 100 ranging from 0.05% to 1% PFA. Please have a look to the attached image.
Can anyone suggest me something to get staining on cell membrane.
Hi Smita,
PFA is a good fixative to see tight and adherent junctions. Alcool and acetone are more for cytoskelet.
You need permeabilization only if the targets of yours antibodies are inside the cells. See antibodies datasheets to verify compatibility with PFA fixation (sometime you need to unmask with PFA fixation).
Pay attention that your wash buffer have to be Mg2+ Ca 2+ supplemented to avoid junction aperture during IHC.
I see your picture, if you want to see mature junction, yours cells have to be at confluence (I think this is the reason why you see protein in cytoplasm).
Good luck,
Virginie.
Thank you Virginie. My washing buffer is DPBS with Ca2+ Mg2+. I used to perform IF at 90% confluent cells. But still I will take in account the concfluency of the cell for this time.
You have to keep in mind that these are not tissue or cell cross-sections, but whole three-dimensional cells on a slide. So if you are staining a protein that is on the cell membrane of a whole cell, you will get staining all over the surface of the cell. In your images you can see that some of the red punctate staining is visible not only over the cytoplasmic areas, but also over the nucleus areas. I would interpret this as being staining of the cell membrane that is overlying the cytoplasm and nucleus, not necessarily inside the cell.
I agree with you Jean-Martin Lapointe. But the staining is not looking like membrane staining even in z Stack mode of microscopy. Also people have used same cells to get junction staining for occludin and they have got nice peripheral ring structure. I did follow exact same protocol as them but I did not get it. I wrote them but I think they were too busy to reply.
This time I will overgrow cells and during imaging I will use z-Stack mode.
Have you already succeeded ? Which cell line do you use ? In case of hCMEC/D3 have you thought about that that these cells may do not form proper tight junctions ?
No still I am struggling for the junction staining. The cell line that I use is HBMEC and it is known in literature that it forms proper tight and adherence junctions. But I am not getting staining for tight as well as adherence junction.
Hi Smita,
Recently, I am start to work with CaCo-2 cell line and for the visualization of the cell-cell contact I use ZO-1 protein, which appears with occludin. Maybe, this marker will be more acceptable for membranous staining.
Dear Smita,
Almost no human cell line form proper tight junctions, also HBMEC could be difficult. Normally you are right, you should find strong signals at the cell-cell contacts. The idea to use ZO-1 is good since this is a tight junction associated Proteins, which is normally easy to stain. The confluency is also a very important part, you have to give the cells time to differentiate after confluency to form proper tight junctions. That means that a shortly confluent layer will need some days of postconfluency to develop their tight junctions. Another issue could be that you antibodies do not work with human cells, in case of tight junction proteins this could be also tricky.
Have you already solved your Problem ?
Hi all,
I also struggle to visualize TJs (with ZO-1 and Claudin-5) in hCMEC/d3 endothelial cells. My images look better than Smita's however I still have intercellular signal and a bit stronger signal on the cell borders. I fixated cells with 2% PFA in PBS and permeablised with 0.5%Tween-20 in PBS. I washed slide with PBS as well. Any sugestions how can I improve my protocol? In case of confluency, I tried different cell concentrations and periods of culturing and I don't think this is the problem...
Thanks in advance,
Magdalena
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