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As I told you on the phone, I am planning on performing IHC on fresh PBMC samples from sheep.
Cells will be isolated from whole blood using a density gradient and will be placed on glass slides using a cytocentrifuge. At this point, we CAN fix them if needed, but we don’t have to. What do you recommend?
Our primary antibody is affinity purified rabbit polyclonal. We care considering using your EXPOSE Rabbit specific HRP-AES detection kit (ab94361) – do you think this is the best kit to use, or would you recommend a different one for our purposes? We are attempting to stain a transmembrane protein.
We are planning to counterstain using hematoxylin: do you think we would get the best contrast with the AEC kit or would you recommend using DAB?
Also, does abcam recommend purchasing hematoxylin from any particular vendor?
If ab64230 the appropriate mounting medium to use with this kit?
Thanks so much for your help. I look forward to your response!
Asked on Jun 20 2012
Thank you for replying with that information.
As I mentioned over the phone, I have spoken to the source of these kits and we do not have any experimental data using either the ACE or DABdetection kits (ab64260, ab64261, ab80437 and ab94361)for IHC on fresh PBMC samples, asallof these kits have only been tested on formalin fixed paraffin embedded tissue samples.Wewould expect that as long as the primary antibody binds the target as expected that both kits could be suitable foryour samples but as we have not tested it ourselves we would not be able to guarentee this.
There should be good contrast if using the AEC or DAB kit with hematoxylin as counterstain. We counterstain with hematoxylin in-house regardless ofwhether DAB or AEC is used. The sensitivity betweenAEC and DABis similar. I would recommend using ab94361 or ab80437 if you expect the PBMC samples to contain any endogenous biotin.I am sorry but I cannot recommend a particular supplier of hematoxylin.
And to answer your final question, the mounting media ab64230 should be suitable for your purposes.
I hope this information has been of help. If you require any further information please do not hesitate to contact us again.
Abcam Scientific Support
Answered on Jun 20 2012