Troubleshooting IHC is a pain. I went through this stuff several months ago and still fight with it.
I'll save you the time and give you antibody-ology in 5 mins. Antibodies have two parts to them. One end is the business end that binds to antigens. The other end is called the Fc portion. Cells such as B cells express receptors to the Fc portion, and they internalize extracellular antibodies and present their associated antigens to T-cells. Cells that express the Fc receptor will often nonspecifically bind antibodies of any species because all Fc portions are really similar. You can buy this stuff called Fc block that should bind up all Fc receptor and prevent it from binding your secondaries. For IHC, the secondary antibodies you buy usually are made in one species to react to another specific species. Let's say you're staining human actin filaments with a goat polyclonal primary antibody. You might use something like a donkey anti-goat IgG as a secondary. To prevent nonspecific binding of the secondary to the tissue, you usually block the tissue with 10% normal serum of the secondary antibody host (in this case, 10% donkey serum)... the idea is that serum has random donkey antibodies that will block any antigen sites that for some reason bind donkey IgG. There are a couple of problems with this. Firstly, serum sometimes doesn't have high enough concentrations of IgG to block things (such as Fc receptor). Secondly, donkey anti-goat reacts with goat but may cross react with all sorts of other animals such as human. If your samples have human IgG on them, this can be a big problem. To prevent this, I always buy antibodies that have been highly cross adsorbed to a whole host of the species to prevent cross reaction. I buy all my secondaries from Jackson Immunoresearch and have been very happy with them, but other companies make them too. I also agree with Jay. Some antibodies are just garbage and will never work despite what the companies say. I always do two things before I buy an antibody. Firstly, I call up the company and ask for literature references. Secondly, I do a literature search for the the clone to see if other people use that antibody and then e-mail them to ask them questions. They are almost always more helpful than the companies. If you're troubleshooting IHC or IF, you should definitely check out the HistoNet listserv. Those people are awesome. Adam On Sun, Jan 10, 2010 at 6:40 PM, Jayakumar, R <[email protected]>wrote: > Welcome to the world of IHC. They only like nice in publications. You got > to keep trying different antibodies before you can get one that works. Every > company claims that their antibodies work, but take that with a pinch of > salt. Usually, they don't. Also check out literature and contact people > how did it well. If you are the first one, tough luck. You just got to > keep trying out different antibodies. Each time, try out different > concentrations, dilutions etc. Everything matters in IHC. If you can > outline the protocol that you are using, that will be a good start. > > Jay > > > > -----Original Message----- > From: [email protected] [mailto: > [email protected]] On Behalf Of Scott Brown > Sent: Sunday, January 10, 2010 5:34 PM > To: [email protected] > Subject: IHC with Nalm-6 > > Hi all, > > I have been trying to do some IHC and IF using Nalm-6 cells, on every > occasion my controls look identical to the one I have used antibody on. > Does anyone know if Nalm-6 cells express surface immunoglobins that my > secondary may be binding to? > > Regards > > Scott > > Scott Brown > PhD Candidate > Molecular Carcinogenesis Program > Children's Cancer Institute of Australia for Medical Research Lowy Cancer > Research Centre, UNSW High Street (PO Box 81) RANDWICK NSW 2031 AUSTRALIA > > Phone: +61 2 9382 1829 > Fax: +61 2 9382 1850 > Email: [email protected] > Web: www.ccia.org.au <http://www.ccia.org.au/> Children's Cancer Institute > Australia is the only independent medical research institute in Australia > solely devoted to research into the causes, prevention and cure of childhood > cancer. Our vision is to save the lives of all children with cancer and > eliminate their suffering. > The information contained in this message and any annexure is confidential > and intended only for the named recipient(s). If you have received this > message in error, please contact the sender immediately and destroy the > original message. > > > > Children's Cancer Institute Australia for Medical Research is the only > independent medical research institute in Australia devoted to research into > the causes, prevention, better treatment and ultimately a cure of childhood > cancer. Our vision is to save the lives of all children with cancer and to > eliminate their suffering. > > The information contained in this message and any annexure is confidential > and intended only for the named recipient(s). 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