ARTICLE

Calcium-induced Modification of Protein Conformation Demonstrated by Immunohistochemistry: What Is the Signal?

Correspondence to: Clive R. Taylor, Dept. of Pathology, U. of Southern California School of Medicine, HMR 204A, 2011 Zonal Ave., Los Angeles, CA 90033.

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl₂ and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl₂ overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl₂ . Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl₂ could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463–469, 1999)

Key Words: Immunohistochemistry, antigen retrieval, protein conformation, chemical modification of, protein structure, antigen–antibody recognition

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				R. W. Sprung Jr., J. W. C. Brock, J. P. Tanksley, M. Li, M. K. Washington, R. J. C. Slebos, and D. C. Liebler Equivalence of Protein Inventories Obtained from Formalin-fixed Paraffin-embedded and Frozen Tissue in Multidimensional Liquid Chromatography-Tandem Mass Spectrometry Shotgun Proteomic Analysis Mol. Cell. Proteomics, August 1, 2009; 8(8): 1988 - 1998. [Abstract] [Full Text] [PDF]

				S. Yamashita and Y. Okada Application of Heat-induced Antigen Retrieval to Aldehyde-fixed Fresh Frozen Sections J. Histochem. Cytochem., November 1, 2005; 53(11): 1421 - 1432. [Abstract] [Full Text] [PDF]

				A R Kranenburg, W I de Boer, V K T Alagappan, P J Sterk, and H S Sharma Enhanced bronchial expression of vascular endothelial growth factor and receptors (Flk-1 and Flt-1) in patients with chronic obstructive pulmonary disease Thorax, February 1, 2005; 60(2): 106 - 113. [Abstract] [Full Text] [PDF]

				S. Yamashita and Y. Okada Mechanisms of Heat-induced Antigen Retrieval: Analyses In Vitro Employing SDS-PAGE and Immunohistochemistry J. Histochem. Cytochem., January 1, 2005; 53(1): 13 - 21. [Abstract] [Full Text] [PDF]

				S.-R. Shi, R. J. Cote, and C. R. Taylor Antigen Retrieval Techniques: Current Perspectives J. Histochem. Cytochem., August 1, 2001; 49(8): 931 - 938. [Abstract] [Full Text] [PDF]

				R. G. Rodrigues, N.-h. Guo, L. Zhou, J. M. Sipes, S. B. Williams, N. S. Templeton, H. R. Gralnick, and D. D. Roberts Conformational Regulation of the Fibronectin Binding and alpha 3beta 1 Integrin-mediated Adhesive Activities of Thrombospondin-1 J. Biol. Chem., July 20, 2001; 276(30): 27913 - 27922. [Abstract] [Full Text] [PDF]

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 1999