Dear colleagues,
I am having troubles with claudin5 and occludin immunocytochemistry in primary human brain microvascular endothelial cells.
I tried different fixatives and permeabilization solutions (acetone/methanol, 4% PFA followed by Triton in PBS etc) and different antibodies (abcam 131259 and invitrogen 35-2500 for cld5, invitrogen 71-1500 and abcam 167161for occludin) at various temperatures for different timepoints.
I also tried to manipulate serum concentrations in the cell medium and cell density ( from serum starved overnight to full serum concentration; from 70% confluent culture to cells kept in culture for 7 days and very tight monolayer).
However nothing seems to work and I cannot get any stain.
I will appreciate if you can share your working protocols and antibody Cat No that work well for human cells as well any any hints / insights of general approach how to properly stain tight junctions in primary cell culture.
Thank you!
Thank you all for the replies!
I did some research and wanted to share some updates that worked for me:
1. It is critical to include Ca2+ and Mg2+ into the washing and fixation solution (to preserve tight junctions integrity).
2. Perform fixation (3% PFA in PBS with Ca/Mg) for 20min at room temperature.
3. Quench PFA residues with 20mM glycine in PBS for 10min RT.
4. Perform permeabilization (0.05% Triton-X in PBS) for 5min at the room temperature.
5. Really high concentration of primary antibodies is needed ( I used Claudin 5 Invitrogen 35-2500 1:20 dilution in 1% BSA blocking buffer). Apply primaries for overnight at 4C.
Here is attached image in Claudine 5 staining in primary human BMVEC
Hello Tetyana,
Sorry to hear that ab131259 isn’t performing as intended. I would suggest to send a note to our scientific support: [email protected]
Thank you - Pierre
Hi Tetyana, I haven't stain my cells, but while doing Western Blot with my cell lysate, I have used the abcam antibodies for claudin 4 and 7. I got good results most of the time. It might be just a simple issue with fixation or cell permeability. I found this article below, might be helpful. Best of luck. Article Visualisation of Multiple Tight Junctional Complexes in Huma...
Thank you all for the replies!
I did some research and wanted to share some updates that worked for me:
1. It is critical to include Ca2+ and Mg2+ into the washing and fixation solution (to preserve tight junctions integrity).
2. Perform fixation (3% PFA in PBS with Ca/Mg) for 20min at room temperature.
3. Quench PFA residues with 20mM glycine in PBS for 10min RT.
4. Perform permeabilization (0.05% Triton-X in PBS) for 5min at the room temperature.
5. Really high concentration of primary antibodies is needed ( I used Claudin 5 Invitrogen 35-2500 1:20 dilution in 1% BSA blocking buffer). Apply primaries for overnight at 4C.
Here is attached image in Claudine 5 staining in primary human BMVEC
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