Julia:
You wrote (two separate messages) I've been given the challenge of finding a variety of fluorochrome conjugated antibodies for IHC, however I'm having a great deal of trouble finding them. We basically want a fluorescent primary antibody that can be visualized instantly without use of a secondary etc. We would like antibodies to bind to Osteocalcin, Alkaline Phosphatase, Periostin, TRAP 5B, Cathepsin-K and N-Cadherin. If anyone has experience using fluorescent antibodies for the above or know of anyone who produces them I would be most grateful if you could let me know. I have recently stained for f4/80 macrophage marker on wax embedded mouse tibia sections. I have managed to get staining however, I also have staining on my negative control, which seems quite specific. I have investigated this and there seems to be no problems with the DAB and all reagents used are exactly the same down to where they are bought as those recommended in a protocol. If you have managed to stain for f4/80 on tibia sections without getting staining on your control or you have any explanations for this I would much appreciate hearing from you to compare protocols. **************************************************************************** **************************************************************************** ****************************************** For fluorochrome conjugated primary antibodies, you can conjugate these on your own. Have you done a search for these antibodies via Google? Be aware the some fluorophore conjugated primaries might not fluoresce IF the antigens are in close proximity to each other. The antibody binds but the fluorochrome itself will not fluoresce due to a physical chemical phenomena called quenching. This is where the fluorophore trades electrons instead, quenching any fluorescence you want to see. If this happens , you would have to detect the fluorophore with antibody, eg. antiFITC, anti Alexa 488 and so on. You are back to square one then. Often it is better to just detect the antibody with a secondary-conjugated to fluorophores, or buy a biotinylated primary antibody and detect with Streptavidin Alexa dye (fluorophore). We do triple IF work for murine CD markers that does not take days to perform, and double IF is very straightforward. As for the F4/80 antibody, you did not provide YOUR protocol so people can respond to the question - fixation, decalcification, retrieval, normal serum blocking, primary and negative control concentrations, etc. This antibody works on FFPE tissue, and there are several postings on Histonet (go to Archives) on protocols for successful staining for soft tissues. The negative control should be negative though. Did you do a dilution panel to determine working concentration of primary? What was secondary? Gayle M. Callis HTL,HT,MT(ASCP) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet