Comparison of different double immunostaining protocols for paraffin embedded liver tissue

Most of the double immunostaining protocols that have been introduced so fa r 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 f urther complicated by tissue-specific difficulties, such as necrosis or hig h 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-an ti-digoxigenin (dig-a-dig/PAP) techniques using different cytokeratin antib odies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitiv ity and specificity in all procedures. Sections, which were stained employi ng PAP/APAP-technique, displayed a higher background staining compared to s ections which were treated with the LAB/LAB or dig-a-dig/PAP protocol. In c ontrast to the dig-a-dig/PAP protocol, the LAB/LAB technique provides a bet ter time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simul taneous detection of different antigens in archived liver tissue.