Possible role of tissue‐bound calcium ions in citrate‐mediated high‐temperature antigen retrieval

Abstract

High-temperature preheating of sections in the presence of a salt (e.g., citrate) or a protein denaturant (e.g., urea) solution has been shown recently to provide a reliable alternative to tissue proteolysis for antigen retrieval from formaldehyde-fixed, paraffin-embedded tissues. However, the underlying mechanism of action of this form of pretreatment remains highly speculative. In this study, we show that calcium chelating agents such EDTA and EGTA are more effective than citrate in the retrieval of a citrate-sensitive nuclear antigen, Ki-67. Also, sodium carbonate and another calcium precipitating agent are both able to effect antigen retrieval at high temperatures. The overall data therefore 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 responsible for the masking of certain antigens.

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

“…Furthermore, the toxic effect of metal salts, particularly lead salts, is a major drawback. Therefore, several alternative AR solutions were subsequently developed, such as citrate, Tris and other buffer solutions, and urea (Kawai et al 1994b;Taylor et al 1994a;Gown et al 1993a;Leong and Milios 1993;Shi et al 1993b;Gerdes et al 1992), EDTA or EGTA (Morgan et al 1994), glycine-HCl buffer (Imam et al 1995), and periodic acid (Kwaspen et al 1995).…”

Protein unlocking procedures of formalin‐fixed paraffin‐embedded tissues: Application to MALDI‐TOF Imaging MS investigations

“…In fact, heating in various solutions has been reported to effectively increase immunostaining of many antigens in routinely processed FFPE material [18]. Several mechanisms have been proposed to explain HIAR such as the disruption of protein-protein crosslinks [18,19] and/or of crosslinks involving calcium ions [20,21] and the improvement of tissue permeability. The positive result of the EDTA treatment seems to agree with the controversial hypothesis on the role of calcium.…”

“…In fact, after reacting with one amino group, formaldehyde can polymerize to form polioxomethylene chains. Calcium ions are able to coordinate to oxygen atoms in this polymer (bonds between the methyl groups of proteins introduced by formaldehyde treatment) and form a cage-like structure which prevent antigenantibody interactions [20,21]. Nevertheless, the effect of calcium ions on immunoreactions might be selective for certain tissue types and not a universal phenomenon [16].…”

Automatic quantification of microvessel density in urinary bladder carcinoma

“…The labelling of vessels was performed as above, except for the AR treatment, where 1 mM EDTA (Morgan et al, 1994), pH 8.0, was used instead of citrate buffer. After developing the first antibody (CD31/CD34) the slides were rinsed in tap water for 10 min before incubation in 1% normal mouse serum (Dako) in PBS for 20 min.…”