Abstract
A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody. Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Torlakovic EE, Nielsen S, Vyberg M et al (2015) Getting controls under control: the time is now for immunohistochemistry. J Clin Pathol 68:879–882
Swanson PE (2015) Immunohistochemistry as a surrogate for molecular testing: a review. Appl Immunohistochem Mol Morphol 23:81–96
Shi SR, Guo J, Cote RJ et al (1999) Sensitivity and detection efficiency of a novel two-step detection system (powervision) for immunohistochemistry. Appl Immunohistochem Mol Morphol 7:201
Skaland I, Nordhus M, Gudlaugsson E et al (2010) Evaluation of 5 different labeled polymer immunohistochemical detection systems. Appl Immunohistochem Mol Morphol 18:90–96
Buchwalow I, Boecker W, Wolf E et al (2013) Signal amplification in immunohistochemistry: loose-jointed deformable heteropolymeric HRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histochem 115:587–594
Larrea MD, Wander SA, Slingerland JM (2009) p27 as Jekyll and Hyde: regulation of cell cycle and cell motility. Cell Cycle 21:3455–3461
Hong F, Larrea MD, Doughty C et al (2008) mTOR-raptor binds and activates SGK1 to regulate p27 phosphorylation. Mol Cell 30:701–711
Matsuda Y, Wakai T, Hirose Y et al (2013) p27 is a critical prognostic biomarker in non-alcoholic steatohepatitis-related hepatocellular carcinoma. Int J Mol Sci 14:23499–23515
Mayer K, Albrecht S, Schaller A (2015) Targeted analysis of protein phosphorylation by 2D electrophoresis. Methods Mol Biol 1306:167–176
Chan A, Matias MA, Farah CS (2011) A novel and practical method using HRP-polymer conjugate and microwave treatment for visualization of 2 antigens raised from the same or different species in paraffin-embedded tissues. Appl Immunohistochem Mol Morphol 19:376–383
Krenacs L, Krenacs T, Stelkovics E et al (2010) Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections. Methods Mol Biol 588:103–119
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741–748
Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol 8:515–520
Morgan JM, Navabi H, Schmid KW et al (1994) Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol 174:301–307
Kalyuzhny AE (ed) (2011) Springer protocols: signal transduction immunohistochemistry, methods and protocols. In: Methods in molecular biology, vol 717. Humana Press, New York
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Grahek, M., Ptak, A., Kalyuzhny, A.E. (2017). High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues Using Secondary Antibody Conjugated to Polymer-HRP. In: Kalyuzhny, A. (eds) Signal Transduction Immunohistochemistry. Methods in Molecular Biology, vol 1554. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6759-9_14
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6759-9_14
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6757-5
Online ISBN: 978-1-4939-6759-9
eBook Packages: Springer Protocols