I don't have an answer for you, but I have observed this with IHC performed on human tissue where Staph is present (primarily skin biopsies). I have seen labeling of Staph organisms with many different types of antibodies (e.g., CD3, CD20, S-100, and more) and I have always attributed it to binding of immunoglobulin to "protein A" in the Staph.
Richard Richard W. Cartun, MS, PhD Director, Histology & Immunopathology Director, Biospecimen Collection Programs Assistant Director, Anatomic Pathology Department of Pathology Hartford Hospital 80 Seymour Street Hartford, CT 06102 (860) 972-1596 (860) 545-2204 ________________________________________ From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Jason Palmer [jpal...@obi.edu.au] Sent: Tuesday, August 26, 2014 3:05 AM To: histonet@lists.utsouthwestern.edu Cc: Phong A Tran Subject: [Histonet] protein A problem with Staph aureus immunostaining Hi all, A question re. immunostaining of bacterial antigens. We are trying to stain cultured MRSA (Staph. aureus) with an antibody to PBP2a, which is the protein that confers antibiotic resistance to these bugs. So far we are finding non-specific binding of our antibodies to protein A is a real problem (or at least we assume that protein A is the problem). Primary is rabbit anti PBP2a and we follow this with a Cy3- conjugated anti rabbit IgG. We have tried normal serum blocking (10% rabbit) before the primary, as well as an Fc fragment of rabbit IgG as a more specific blocker of protein A before the primary, but so far without much success; we see labe ling with our primary, with an isotype control and also with diluent alone. So it would seem that both our primary and secondary is binding to protein A non-specifically. Next things to try are increasing the concentration of the Fc fragment blocking (was about 100ug/ml the first time) and also to try an Fab fragment as a secondary, rather than the whole IgG we currently use. Does anybody have any other suggestions as to what we might try to get on top of this problem? I have only ever immunostained mammalian cells and tissues before, so this is my first time with bacteria. The dyes I'm using (Cy3 and DAPI) photobleach very easily for some reason with these bugs compared with your average mammalian cells? Thanks for any suggestions, Jason Jason Palmer Histology Laboratory Coordinator O'Brien Institute 42 Fitzroy St, Fitzroy Victoria 3065 Australia tel +61 3 9288 4045 fax +61 3 9416 0926 email: jpal...@obi.edu.au _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure, or distribution is prohibited. If you are not the intended recipient, or an employee or agent responsible for delivering the message to the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message, including any attachments. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
