Hi you can try Abcam antibody is work well in human sample and  also find with mouse origin in same company . your following  immunoblot porocol ok, incubate secondary antibody 90 min or longer you will get good signal and wash 3 time with TBST buffer (10min x3 )each wash at least 10 min or if have any background wash more.

We have been quite happy with Abcams Cld-5 AB's, you could try out http://www.abcam.com/claudin-5-antibody-ab15106.html or similar.

try to increase secondary, especially if you do not have background. i assume your target is a minor protein in your samples.

Dear Ravichandran Panchanathan and Birger Brodin

I'm apreciated for your suggestion to use abcam cldn-5 Ab, I'll try it ! 

 Dear Attila Lehotzky

Thank you for your suggestion. claudin-5  is expressed in HUVEC, but very low as you pointed out. Further more, antibodies I used had many non-specific bands, and these bands signals were stlonger than that of claudin-5, so the band signal of claudin-5 was hard to detect. I'll modified with secondary Ab dilution and incubation time.

Dear Kyohei,

Is Caudin-5 easily detectable in HUVEC by immunocytochemistry? I have to assess the expression of this protein in HUVEC monolayer but under the microscope, whole of the cells are green although I washed them after staining with Antibody 3 times. could you please et me know which protocol you follow for Claudin-5 in HUVEC? Thanks, Nasim

Dear Nasim

 Thank you for your request about immunostaining of claudin-5 in HUVEC.  Staining protocol I use is described above,  and cell culture protocol is below: 

 HUVECs are seeded at 100,000 cells/cm2 and cultured on type I collagen-coated 24-well Transwell® inserts. Type I collagen, Cellmatrix® was purchased from Nitta Gelatin, Osaka, Japan. In this cell concentration, HUVEC will be cultured confluently after 24h. After 48 h, medium are exchanged, and HUVEC are incubated further 72h to get stable in permeability of HUVEC. after that I use in each experiment. So, I use HUVEC in immunostaining after 5-7 days from seeding. Is your protocol too fast to express claudin-5?  

Dear Kyohei,

Thank you for your email, I seeded cells at the density of 5000 /cm2 in 24 well plates on glass coverslips coated with fibronectin on day 5 and afterwards, I started immunolabeling, the Antibody is conjugated with FITC, So I only use primary antibody and there is no secondary, is it necessary to permeabilize cells for fixation ? which method of fixation is better for immunocytochemistry?methanol or paraformaldehyde for HUVEC in order to detect Claudin-5?

Claudin 5 is expressed as an oligomer, as a dimer or larger, rather than as a monomer.  The 23kD weight noted by most companies assumes that you've used lysis and reducing conditions sufficient to break apart the higher MW oligomers into monomers, but at least in my experience, this isn't always the case.  I regularly get dimers in human samples and up to tetramers and sometimes even higher with mouse brain samples.  Dimers for Claudin 5 run at about 37, while tetramers are about 60-65 and quads are about 100 or so.  Your higher MW "non-specific bands" are likely your target, but without seeing an image, I can't say for certain.