An enhanced immunocytochemical method for staining bone marrow trephine sections.

Abstract

Aims-The detection of cellular antigens in fixed decalcified bone marrow trephine (BMT) sections depends on the method of processing, the nature of the antigen and antibody, antigen retrieval techniques, and the sensitivity of the immunocytochemical method. This study evaluated a tyramide enhanced avidin-biotin inmunostaining method on formalin fixed decalcified BMT sections to determine whether the method could detect previously undetectable antigens. Methods-Nineteen BMT biopsies from a range of haematological disorders were evaluated with 43 antibodies to haemopoietic antigens using horseradish peroxidase and alkaline phosphatase detection methods, using the tyramide enhanced avidin-biotin immunostaining method. Results-Compared with standard avidin-biotin immunostaining methods the tyramide enhanced immunostaining method showed enhanced signal intensity, gave positive labelling for antigens that require pretreatment by other methods, and previously unreactive antigens were detected. Primary antibodies could be used at up to 200 times higher dilutions. Conclusion-The tyramide enhanced immunostaining method, while retaining specificity, is highly sensitive and enables an increased number and range of antigens to be detected than previously possible. The method could be applied to BMT sections for the routine diagnosis and classification of haematological disorders.

Novel Application of Tyramide Signal Amplification (TSA): Ultrastructural Visualization of Double-labeled Immunofluorescent Axonal Profiles

“…Essentially, in the TSA technique the biotinor fluorophore-conjugated tyramide is "attracted" to the antigenic site activated by the peroxidase component of the immunolabeling complex. The activated tyramide instantly and covalently binds to nearby tyrosine molecules, and this single enzymatic reaction greatly (1-400-fold) amplifies the signal intensity of the immunocytochemistry (Bobrow et al 1989(Bobrow et al ,1992Hunyady et al 1996;Erber et al 1997;van Gijlswijk et al 1997). Soon after the technique was first described, its widespread utilization (Van Heusden et al 1997;Kaufmann et al 1998;Kressel 1998;van de Corput et al 1998;Speel et al 1998Speel et al ,1999 and further modifications in its application highlighted its usefulness and importance, particularly in paradigms involving fluorescent double labeling with antibodies derived from the same species (Hunyady et al 1996;Shindler and Roth 1996;Teramoto et al 1998).…”

The expression of antiapoptotic proteins Bcl-2, Bcl-XL, and Mcl-1 in benign, dysplastic, and malignant biliary epithelium

“…This method also incorporates streptavidin-biotin complex, but has additional steps made up of biotinylated tyramide signal enhancement (recommended by Dako). 35 The system was adapted for rabbit primary antibody using the biotinylated antirabbit link (Dako). Antihuman Mcl-1 (Dako) and Bcl-X L (Autogen Bioclear, Calne, Wiltshire, UK) primary polyclonal antibodies were incubated with slides through a series of 15 minute steps at room temperature at a dilution of 1/1000 and 1/750, respectively.…”

DNAse I pre-treatment markedly enhances detection of nuclear cyclin-dependent kinase inhibitor p57Kip2 and BrdU double immunostaining in embryonic rat brain

“…Following four PBS rinses, antigen was visualized using Xuorescein-avidin D (1:2,000) according to manufacturer's instructions (Vector Lab). The use of biotinyl tyramide ampliWcation to enhance biotin-labeled immunohistochemical sites has been described extensively (de Haas et al 1996;Hunyady et al 1996;Erber et al 1997;Plenat et al 1997;Kressel 1998;van Gijssel et al 1998). Following PBS rinses, sections were mounted using ProLong Gold antifade reagent (Molecular Probes) for images analysis.…”