Quenching autofluorescence in tissue immunofluorescence

Background: Immunofluorescence (IF) is one of the most important techniques where fluorochromes conjugated to antibodies are used to detect specific proteins or antigens. In tissue sections, autofluorescence (AF) can lead to poor quality images that impair assessment. The placenta is a pivotal extra-embryonic organ in embryo development, where trophoblasts make up a large proportion of the cells. Teratoma formation is one of the critical assays for pluripotent stem cells. Methods: We tested whether ultraviolet (UV), ammonia (NH3), copper (II) sulfate (CuSO4), Trypan Blue (TB), Sudan Black B (SB), TrueBlackTM Lipofusin Autofluorescence Quencher (TLAQ) and combinations of these treatments could reduce AF in paraffin and frozen sections of placenta and teratoma in FITC, Texas Red and Cy5.5 channels. Results: We found that UV, NH3, TB and CuSO4 quenched AF to some extent in different tissue and filters, but increased AF in Texas Red or Cy5.5 channels in some cases. SB and TLQA exhibited the most consistent effects on decreasing AF, though TLQA reduced the overall IF signal in placenta sections. Not all combined treatments further reduced AF in both placenta and teratoma sections. Conclusions: SB and TLAQ can effectively quench AF in placenta and teratoma IF. 1 1 1 2 2 1