Abstract 2734: Click colorimetric EdU proliferation and TUNEL assay: Conversion of click chemistry from fluorescent to colorimetric for use with bright field microscopy

Abstract

Abstract We present here the conversion of click chemistry fluorescence-based EdU proliferation assay and TUNEL apoptosis assay to use with bright field microscopy. Advantages of having a colorimetric signal are the retention of the click chemistry work flow while allowing for compatibility with standard H&E staining and protocols. Proliferation using copper-catalyzed labeling of the thymidine analog EdU (ethynyl deoxyuridine) was introduced in 2007. This click-based assay has advantages over the traditional antibody based BrdU assay. The small molecule detection is simple, rapid and gives a bright signal useful for multiplexing. Additionally, recent improvements to the chemistry allow for GFP and R-PE compatibly with the EdU and TUNEL assays. Several approaches for creating a colorimetric signal for EdU proliferation were attempted. The method presented here had the shortest protocol and uses a biotin-based click reaction followed by streptavidin -horseradish peroxidase (HRP) step. This two-step reaction creates an essentially covalent link between the EdU incorporated in the DNA and the enzyme reporter. Then standard HRP chromogens such as DAB were used to produce a signal marking the proliferating cells. Examples of proliferation using colorimetric EdU labeling in adult Zebrafish caudal fin, mouse cardiac, and rat intestine, mammary and uterine FFPE treated tissue are presented. Multiplexing with alkaline phosphatase for two color detection are also presented. Two color detection of EdU with BrdU or EdU with progesterone receptor (PR) are shown. Most HIER protocols are compatible with EdU detection. For EdU alone no HCl treatment is required. For EdU/BrdU double labeling the BrdU protocol for DNA revelation is used. Anti-BrdU mouse antibody clone MoBU-1 is used because of its lack of cross-reactivity with EdU. Additionally, we present the click chemistry-based apoptotic TUNEL assay colorimetric detection using the same approach. Clickable biotin adapters are used to couple HRP to the ethynyl tails created by TdT enzyme extension at 3′ end of double stranded breaks. We provide examples of this late stage indicator of apoptosis visualized with DAB substrate and stained with standard H&E and other histological stains. Citation Format: Scott T. Clarke, Carmen Finnessy, Fernada M. Bosada, Ben Armstrong, Michael O’Grady. Click colorimetric EdU proliferation and TUNEL assay: Conversion of click chemistry from fluorescent to colorimetric for use with bright field microscopy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2734.