Electrochemical antigen-retrieval of formaldehyde fixed and paraffin-embedded archived leprosy skin biopsies

Abstract

While formaldehyde fixation preserves tissue morphology, it often hinders immunodetection of antigens in paraffin-embedded tissue because the antigens are masked. Antigen unmasking can be achieved with treatments such as microwave irradiation but they often lead to excessive tissue damage. Therefore, an electrochemical antigen-retrieval method (EAR) was devised in which an alternating electric current is passed through the tissue in a chamber containing an electrolyte buffer. The results obtained with this method were compared to those after microwave irradiation using archived samples of formaldehyde-fixed and paraffin-embedded lepromatous leprosy skin. The efficacy of the two unmasking procedures was assessed by the immunodetectability of several marker antigens using 24 antibodies. Fifteen antibodies that were directed against transmembrane proteins (CD), and the remaining 9 against cytokeratins 18.6 and 19, laminin, vimentin, S100a, BCG, Ulex europaeus lectin, PCNA, and P21ras. Simple and double immunohistochemistry was performed using the universal ENVISION and LSAB + AP detection systems. After unmasking with the EAR method, immunoreactivity was clearly detected with 22 of the 24 antibodies in single labeling reactions. They include the critical antigens CD3 and CD4 for identifying the T lymphocyte lineages. In contrast, only 20 of the antibodies reacted after microwave irradiation. After double immunolabeling, immunoreactivity was quantitatively similar with both methods. However, the EAR unmasking produced a stronger labeling reaction. Thus, with double labeling immunohistochemistry, EAR made it possible to use higher antibody dilutions and shorter incubation times. Heat damage was also prevented. In conclusion, EAR treatment produces better staining results than microwave irradiation treatment.