[Establishment of multiple-indirect immunofluorescence technique for detecting multiple markers in the same specimens of hepatoma cells and frozen tissues].

Objective To establish a multiple-indirect immunofluorescence staining that can be used to detect several proteins in the same frozen section of hepatocellular carcinoma (HCC) tissues. Methods Laser scanning confocal microscopy (LSCM) was applied to scan the first group of antibodies against four proteins stained on the HCC tissues by the indirect immunofluorescence technique. The first group of antibodies and residual dye were removed by homologous species IgG Fab antibody blocking combined with chemical strip reagents (2-mercaptoethanol/dodecyl benzene sulfonic acid sodium/guanidine hydrochloride/sodium borohydride, β-ME/SDS/GnHCl/NaBH4). The second group of antibodies against four proteins were stained in the same section and imaged with LSCM again. Results The antibodies against eight proteins were co-stained and observed in the same section by LSCM. The antibodies of IgG Fab could close non-specific dying. After the first round of residual antibody was cleared by strip reagents, the second round of antibodies was successfully stained in the same section. Conclusion The experiment established the multiple-indirect immunofluorescence techniques for detecting multiple markers in the same tumor tissues as expected.