2011-2012 Catalogue - PeproTech, Inc.

PEPROTECH ®www.peprotech.comtors which influence the optimization of the fixationprocess include the purity, affinity and specificity ofthe detecting antibody, the structure and stability of thetarget protein, and the structural and biochemical characteristicsof the tissue sample.The fixation process, while critical for maintainingthe integrity of the tissue, often masks the antibodyrecognition epitope on the target protein by inducingchemical modification, conformational alterations, orprotein aggregation. Therefore, most IHC procedurescontain an unmasking step, which is frequently called“antigen retrieval” or “epitope recovery”. This unmaskingprocess can be accomplished by several procedures,including HIER (Heat-Induced Epitope Retrieval) andPIER (Protease-Induced Epitope Retrieval), which,depending on the sample and the target protein, can beused alone or in tandem.The detection strategies commonly applied in IHCprocedures can be classified into two general categories,designated as direct and indirect detection methods.The direct detection methods utilize a labeled an-tibody, capable of fluorescing or activating a chemicalchromogen, which binds directly to the target protein.This method has the advantages of simplicity, low nonspecificbinding in most cases, and short assay “runtimes.” Indirect methods use an unlabeled primary antibodyto initially bind the target protein, which is thenrecognized by a labeled secondary antibody. Some indirectIHC detection procedures use a “three stage” procedure,in which an unlabeled secondary antibody isused and subsequently recognized by a labeled tertiaryantibody. Most indirect methods produce some degreeof signal amplification during the multiple stages of thedetection procedure, which consequently results in increasedassay sensitivity.Figure V-1 is a sample IHC detection protocolrepresenting the use of an “indirect immunoenzymatic”method.References(1) Boenisch, Thomas ed. Immunochemical staining procedureshandbook. 3 rd ed. Carpinteria, CA; 2001.(2) Janeway, et al. Immunobiology: the immune system in healthand disease. 6 th ed. New York: Garland Science, 2005.INFORMATION & TECHNICAL SUPPORTGoat anti-human VEGFHuman Breast Ductal Carcinoma immunochemically stainedwith Goat anti-human VEGF (Catalogue #500-P10G) using aHorseradish Peroxidase-DAB (brown) detection system withhematoxylin (blue) counterstain.Dark brown areas indicate stained carcinoma tumor cells.Light brown areas indicate stained duct and vessel lining.Rabbit anti-human IL-6Human Cervical Squamous Cell carcinomaimmunochemically stained with Rabbit anti-human IL-6(Catalogue #500-P26) using an Alkaline Phosphatasepolymer enhanced detection system with Permanent Red andhematoxylin (blue) counterstain.The cytoplasm of the carcinoma cells is stained red.Bestellungen: Deutschland: Tel: 0800 436 99 10 • email: info@peprotech.dePour commander: France: Tel: +33 (0)1 46 24 58 20 • email: info@peprotechfr.comTo Order: NORDIC: Tel: +46 (0)8 640 41 07 • email: info@peprotech.se173