Is the Catalyzed System Amplification (CSA) II Kit Also Applicable for Cryostat Tissue Sections and Double Staining?

Abstract

Commercial tyramide-detection kits for immunohistochemistry are developed for the extremely sensitive visualization of low abundant antigens/epitopes in formalin-fixed and paraffin-embedded tissue sections. Most kits are based on biotin–streptavidin interaction and therefore known with many incubation steps and possibly unwanted staining of endogenous biotin. The recently introduced catalyzed system amplification II kit (CSA II) is unknown with these drawbacks and is tested here for an application on acetonefixed cryostat tissue sections as well as for an immunoenzymatic double staining using two unlabeled mouse primary antibodies. Because commercial tyramide detection kits are developed for the staining of formalin-fixed and paraffinembedded tissue sections, detection on cryostat tissue sections yields an overstained specific image along with moderate nonspecific background staining that is also present in blank sections without primary antibody. This article will show that, for cryostat tissue sections, dilution of the tyramide kit components yields a strong and specific staining with antibodies that hardly showed any specific staining when detected with a regular 3-step streptavidin-biotin visualization procedure. The T-cell activation markers CD40Ligand and CD69 serve as model system. Double staining using two unlabeled mouse primary antibodies of the same isotype is usually a problem. The CSA II kit is tested here in a double-staining procedure in which a very high dilution of the first primary antibody is followed by the highly sensitive CSA I1 detection. Subsequently, a rather low-sensitive two-step alkaline phosphatase staining is applied. Because the first primary antibody is applied in a very high dilution, eventual cross reaction of anti-mouse from the second detection system with the first mouse primary antibody remains below detection level. This double staining procedure offers a great tool for both visualizing two different cell populations and colocalization by mixedcolors. (The J Histotechnol 29:157, 2006)Submitted February 20, 2006; accepted June 26, 2006