I is my understanding that the Mach 3 is for use on human clinical samples and will not work in mouse tissue. Maybe BioCare technical person didn't realize you were using human cell in the mouse. BioCare has a great polymer for rabbit antibodies on rodent tissue in their ProMark series. "Rabbit on Rodent HRP Polymer" cat RMR622, also comes in AP format RMR625. This is a great product especially when signal is low. Great amplification and no background. I am not aware of the Starr Trek system, going to have to check it out.
------------------------------ Message: 10 Date: Mon, 2 May 2011 11:46:29 -0500 From: "Troutman, Kenneth A" <ashley.trout...@vanderbilt.edu> Subject: Re: [Histonet] Tumors tumors everywhere To: "Histonet@lists.utsouthwestern.edu" <Histonet@lists.utsouthwestern.edu> Message-ID: <7b310892042da74cb3590053f424cfe613d63cd...@its-hcwnem06.ds.vanderbilt.edu> Content-Type: text/plain; charset="us-ascii" Hi Sarah, Biocare's MACH3 is designed to work with mouse or rabbit antibodies, so it is likely picking up some mouse antigens: http://biocare.net/products/detection/mach-3/ MACH 3(tm) MACH 3 is a two-step, biotin-free detection system which provides excellent specificity, sensitivity and nuclear staining for mouse or rabbit primary antibodies. I would choose a reagent that recognizes ONLY your rabbit antibody. Check out Biocare's Starr Trek components (like the Trekkie Rabbit link. I know the name is goofy... :)). They should be able to help you. Good luck. Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 Message: 7 Date: Thu, 28 Apr 2011 15:16:16 -0500 From: <sgoe...@mirnarx.com> Subject: [Histonet] Tumors tumors everywhere To: <histonet@lists.utsouthwestern.edu> Message-ID: <d957f2a7d21959488c492a2680f9920a224...@svrexch.asuragen.us> Content-Type: text/plain; charset="us-ascii" So I am staining tumors that were implanted as cells subdermally into mice. The cells are human. I am trying to do Caspase staining on these tumors. The primary is an anti-human rabbit polyclonal, and I am using a polymer (Biocare Mach3) in lieu of the secondary antibody. The background is through the roof!! Could the reason be that the tumor was grown in a mouse and is having cross reactivity somehow? What species antibody should I be using instead? All my mouse monoclonal antibodies work perfect on the tissue, it's this stupid rabbit polyclonal!! I am blocking endogenous enzymes (peroxidase etc., DAKO), avidin and biotin (just to see if that would help...it didn't), and protein block (it's literally an hour worth of blocking!!), developing with DAB (Dako) and hematoxylin counterstain. I am so confused as how to get this to work! Also, it isn't just this particular antibody it is any rabbit polyclonal I have tried. Could it be the polymer? It is the one that Biocare suggested? HELP!! Thanks in advance =) Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet