Hi,
I am trying to staining for brain vessels using claudin-5 antibody (Invitrogen, 35-2500). I tried many times and this antibody still do not work on my hands. The way I did was: perfuse mice with 4% PFA, then post-fix in 4% PFA 4 degree O/N, then dehydrate in 30% sucrose for 1-2 days, after sectioning it into 30um sections using cryostat, the sections were staining using floating staining method. I tried 1:400 and 1:1000 dilution. Both did not work. I wonder if anyone can help me troubleshoot this problem. Any help will be grateful.
Please look at the following below link which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292788/pdf/nihms359020.pdf
Thanks
Try increasing the concentration. Usually, when I get a new antibody I do three different concentrations:
1) 5-10 times lower
2) Recommended
3) 5-10 times higher
Sometimes it might require a little bit more playing.
The final results will also depend on the secondary you are using. If you are using Alexa Flour try 1:500, it works the best for my samples. For other secondaries, you might need higher concentrations. Also, try not to over fix the samples.
All the best
The claudin-5 antibody (Invitrogen, 35-2500) does not work well in PFA-fixed tissue. Do not perfuse with PFA, directly freeze the brains on dry ice and make cryosections. Use methanol for fixation of the sections.
Divine C Nwafor I usually work with 1:200-1:500 dilution of the antibody.
Steffen Storck Thanks hope to see you at the Barriers of CNS GRC/GRS in june!
- Badges
- Science topic
- Similar topics
- Geoscience
- Atmospheric Sciences
- Atmosphere