ABSTRACT
RNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously.
Subject(s)
Diagnostic Imaging , Immunohistochemistry , Nucleic Acid Hybridization , RNA, Messenger/genetics , Animals , Embryo, Mammalian , Embryo, Nonmammalian , Humans , In Situ Hybridization , In Situ Hybridization, Fluorescence , RNA, Messenger/isolation & purification , ZebrafishABSTRACT
We present the development of a three-dimensional (3-D) combinatorial cell culture array device featured with integrated three-input, eight-output combinatorial mixer and cell culture chambers. The device is designed for cell-based screening of multiple compounds simultaneously on a microfluidic platform. The final assembled device is composed of a porous membrane integrated in between a Parylene 3-D microfluidic chip and a PDMS microfluidic chip. The membrane turned the cell culture chambers into two-level configuration to facilitate cell loading and to maintain cells in a diffusion dominated space during device operation. Experimentally, we first characterized the combined compound concentration profile at each chamber using a fluorescence method. We then successfully demonstrated the functionality of the quantitative cell-based assay by culturing B35 rat neuronal cells on this device and screening the ability of three compounds (1,5-dihydroxyisoquinoline, deferoxamine, and 3-aminobenzoic acid) to attenuate cell death caused by cytotoxic hydrogen peroxide. In another experiment, we assayed for the combinatorial effects of three chemotherapeutic compound exposures (vinorelbine, paclitaxel, and γ-linolenic acid) on human breast cancer cells, MDA-MB-231. The same technology will enable the construction of inexpensive lab-on-a-chip devices with high-input combinatorial mixer for performing high-throughput cell-based assay and highly parallel and combinatorial chemical or biochemical reactions.