Optimal autofluorescence blocking for reliable immunofluorescence in human ovarian samples: A comparison of 4 blocking kits.

INTRODUCTION: Fertility depends on the complex process of folliculogenesis, yet many aspects of early human follicle development remain unclear. Immunohistofluorescence (IHF) enables detailed analysis of follicular structures and allows quantification. However, it is often affected by tissue autofluorescence. This study compares four commercially available kits to identify the most effective method for reducing autofluorescence and improving IHF accuracy. METHODS: Sections of human ovarian tissue were treated according to the staining protocol without antibodies, but with one of four kits reducing autofluorescence to compare their efficacy. Images were taken on the Ti2-E fluorescence microscope in three different fluorescence channels.The kit with the highest reduction in autofluorescence was applied for a strong and a rather subtle staining in order to analyze the signal-to-background ratio (SBR). RESULTS: All tested reagents reduced tissue autofluorescence in all three fluorescence channels. Among the evaluated products, TrueBlack showed the greatest reduction in autofluorescence. When combined with IHF staining, it significantly reduced background fluorescence and improved the SBR. Nevertheless, TrueVIEW and Ready Probes were similarly effective. CONCLUSION: Autofluorescence in human ovarian tissue can be effectively reduced using commercially available reagents. Among the tested products, TrueBlack showed the highest efficacy with our specific protocol and setup. Given the high demand for research and the rarity of suitable human ovarian samples, sharing these results may support more accurate and reproducible immunofluorescence analyses in future studies.