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Thank you for your reply, that is very interesting. We had selected to use CD51/61 as a marker for angiogenesis, we are looking into the pathological outcome of possible angiogenesis in brain tissue. We had selected the glioblastoma case as a positive control to test the antibody before running it through our cohort. Do you have any recommendations of how we can increase specific staining of the antibody? Do you believe then that staining of blood is just monocytes, we had suspected that it maybe red blood cells.
I look forward to hearing your response
Asked on Apr 03 2012
Thank you for your email.
The sensitivity of staining could be increased by;
- using enhancers; we have DAB enhancer in catalogue, please check the datasheet https://www.abcam.com/DAB-Enhancer-ab675.html
- Using lower dilution of primary or secondary abs without compromising the background
- Incubating primary overnight at 4C and secondary for 2 hours RT
- Ab Optimization; In IHC-P optimization of antibodies is always necessary for finding the right dilution and best antigen retrieval procedure e.g. sometime 5 min microwave give clean staining than 10 min microwave. In my own experience microwave 10 minutes gave cleaner staining than 5 minutes so it is good to check which experimental condition is best suitable in relation to tissue section and antibodies.
RBCs do not express CD51 and CD61 so the antibody should not bind to these cells. To confirm this you may consider dipping the slides in hematoxylin solution, RBCs do not have nucleus so they will not be stained.
The following website is a also a good resource for searching the endothelial targets; http://www.antibodybeyond.com/reviews/tumor-markers/angiogenesis-marker.htm
Let me know what you think.
I hope this information will be helpful. Should you have any question please do not hesitate to contact me.
Answered on Apr 03 2012