Comparison of different double immunostaining protocols for paraffin embedded liver tissue.

Most of the double immunostaining protocols that have been introduced so far have been developed for application on fresh frozen material or based on different species antibodies. In liver tissue, general problems of double immunostaining techniques are further complicated by tissue-specific difficulties, such as necrosis or high intracellular protein content. To assess a reliable double immunostaining protocol for archived, paraffin embedded liver tissue, different protocols based on the use of same species primary antibodies were evaluated in terms of sensitivity, specificity and non-specific background staining in pathological liver specimens. We compared peroxidase-anti-peroxidase, alkaline phosphatase-anti-alkaline phosphatase (PAP/APAP), labelled-avidin-biotin (LAB/LAB) and digoxigenin-anti-digoxigenin (dig-a-dig/PAP) techniques using different cytokeratin antibodies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitivity and specificity in all procedures. Sections, which were stained employing PAP/APAP-technique, displayed a higher background staining compared to sections which were treated with the LAB/LAB or dig-a-dig/PAP protocol. In contrast to the dig-a-dig/PAP protocol, the LAB/LAB technique provides a better time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simultaneous detection of different antigens in archived liver tissue.