Semiautomated multiplexed quantum dot-based in situ hybridization and spectral deconvolution - PubMed (original) (raw)

Figure 1

Use of direct quantum dot oligonucleotide conjugates for in situ detection of mcc10 in mouse lung. A: i, Agarose gel (0.8%) electrophoresis showing no migration of unconjugated streptavidin-coated QDs (lane 1) and a differential gel shift with increasing probe-to-QD ratio. The individual samples are, from left to right, 605-nm streptavidin-coated QD alone (lane 1) followed by molar ratios of QDs to probe of 5:1; 1:1, and 1:10 (lanes 2 to 4). ii, Agarose gel (0.8%) electrophoresis showing strongest migration of unconjugated amine coated QDs (lane 1) and reducing gel shift with increasing probe-to-QD ratio. The individual samples are, from left to right, 580-nm amine coated QD alone (lane 1) followed by molar ratios of QDs to probe: 1:20, 1:10, 1:5, 1:2, 1:1, 2:1, and 5:1 (lanes 2 to 8). B: Formalin-fixed, paraffin-embedded mouse (H&E ×20 shown in i) lung hybridized with DIG-labeled antisense mcc10 riboprobe (ii) showing localization of signal to Clara cells located in the bronchiolar epithelium. Magnification, ×20. iii, DIG-labeled mcc10 antisense oligonucleotide showing similar expression distribution to that observed with the antisense mcc10 riboprobe. Magnification, ×20. iv, 5′-biotinylated mcc10 antisense oligonucleotide (sequence the same as in iii) in mouse lung disclosed using a 605-nm streptavidin-coated QD, demonstrating strong signal and localization to the bronchiolar epithelium. Magnification, ×20. v, DIG-labeled antisense mcc10 riboprobe disclosed with a 655-nm QD, demonstrating high signal in the same distribution to that of the QD-disclosed oligonucleotide probe (iv). Magnification, ×20. vi, IHC for mcc10 disclosed with diaminobenzidine demonstrated strong cytoplasmic staining in the same distribution to that seen with either riboprobes or oligonucleotide probes (ii–v). Magnification, ×20. C: i, Formalin-fixed, paraffin-embedded wild-type mouse fibroblasts hybridized with DIG-tailed antisense cyclin E1 oligonucleotide probe showing strong hybridization signal [4,6-diamidino-2-phenylindole (DAPI) counterstained]. Magnification, ×40. ii, Formalin-fixed, paraffin-embedded cyclin E1 knockout mouse fibroblasts hybridized with DIG-tailed antisense cyclin E1 oligonucleotide probe showing the absence of hybridization signal (DAPI counterstained); small amounts of particulate QD are present, but cytoplasmic signal is absent. Magnification, ×40.