POSSIBLE ROLE OF TISSUE-BOUND CALCIUM - IONS IN CITRATE-MEDIATED HIGH-TEMPERATURE ANTIGEN RETRIEVAL

High-temperature preheating of sections in the presence of a salt (e.g ., citrate) or a protein denaturant (e.g., urea) solution has been sho wn recently to provide a reliable alternative to tissue proteolysis fo r antigen retrieval from formaldehyde-fixed, paraffin-embedded tissues . However, the underlying mechanism of action of this form of pretreat ment remains highly speculative. In this study, we show that calcium c helating agents such EDTA and EGTA are more effective than citrate in the retrieval of a citrate-sensitive nuclear antigen, Ki-67. Also, sod ium carbonate and another calcium precipitating agent are both able to effect antigen retrieval at high temperatures. The overall data there fore suggest that either the chelation or the precipitation of tissue- bound calcium ions, and perhaps also other divalent metal cations, is a critical step in salt-mediated antigen retrieval. As a corollary, it is suggested that tight complexing of calcium ions or other divalent metal cations with proteins during formaldehyde tissue fixation is res ponsible for the masking of certain antigens.