Fig 1: Specific DGC marking and imaging of CTGF in AD brain of 3-month-old APP/PS1 mice in vivo.a The in vivo NIR-II fluorescence images of wild-type (WT) mice (left) and 3-month-old APP/PS1 mice (right) after intravenous injection of DGC. And the ex vivo imaging of brains isolated from mice (cardiac perfusion with saline) after DGC injection (λ ex = 808 nm). The experiment was repeated in three independent pair of mice with similar results. b In situ fluorescence (red) imaging, LA-ICP-TOF-MS imaging, and DAB chromogenic (brown) imaging of CTGF in brain sections prepared from the ex vivo NIR-II analyzed brains. Inset pictures are enlarged areas of DAB chromogenic imaging. Scale bar = 50 μm. Each experiment was repeated independently for three times with similar results. c The brain sections prepared from the brains after ex vivo NIR-II imaging, sections were stained with FITC-labeled CTGF antibody (green fluorescence). Scale bar = 20 μm. The inset showed the colocalization of DGC (red fluorescence) and CTGF antibody (green fluorescence). Scale bar = 20 μm. The experiment was repeated independently in three pairs of mice with similar results. d Representative images of DGC (red fluorescence) location in cells of brain sections. GFAP marked reactive astrocytes, CD31 marked vascular endothelial cells, Neun marked neurons, and Iba 1 marked microglia cells (all green fluorescence). Scale bar = 20 μm. The inset showed the colocalization of DGC (red fluorescence) and CTGF (green fluorescence) expressed by reactive astrocytes and vascular endothelial cells rather than microglia cells and neurons. Each experiment was repeated independently three times with similar results.
Fig 2: DGC identifies early-stage AD patient brain sections.a The fluorescence imaging of cortex regions in brain sections from an early stage of AD patient (AD) and Healthy control (HC) after staining with DGC (red fluorescence) and FITC-labeled CTGF antibody (green fluorescence). The merge pictures show the colocalization of DGC and CTGF. Scale bar = 50 μm. b The fluorescence (FL) intensity of DGC in (a) analyzed by ImageJ software. Data were presented as mean ± SD from three independent sections of the same case (n = 3). Student’s t-test. c ICP-MS analysis of DGC (Au) content of per cm2 after incubating DGC with indicated brain sections. Data were presented as mean ± SD from three independent sections of the same case (n = 3). Student’s t-test. Source data are provided as a Source Data file.
Fig 3: Specific DGC marking and imaging of CTGF in AD patient-derived brain sections and healthy control (HC) brain sections.a The fluorescence imaging of hippocampal and cortex regions in brain sections from AD patient and HC after staining with DGC (red fluorescence) and FITC-labeled-Aβ antibody (green fluorescence). The merge pictures show the partial colocalization of DGC and Aβ. Scale bar = 20 μm. b The fluorescence imaging of hippocampal and cortex regions in brain sections from AD patient and HC after staining with DGC (red fluorescence) and FITC-labeled CTGF antibody (green fluorescence). The merge pictures indicate the colocalization of DGC and CTGF. Scale bar = 20 μm. c Representative chromogenic imaging (brown) of hippocampal and cortex regions in brain sections from AD patient and HC after staining with DGC and DAB working solution. Scale bar = 20 μm. Each experiment was repeated independently in two AD patient-derived brain sections and two HC brain sections with similar results.
Fig 4: The affinity and specificity of DGC for targeting CTGF.a SPR data indicates the binding kinetics of free DAG (left) and DGC (right) with CTGF. The KD value of DAG is 22.3 μM and that of DGC is 21.9 nM. b Immunoblotting of CTGF was performed in U87MG, SH-SY5Y and CTX TNA2 cells. The CTGF expression levels are analyzed by imageJ software. Data were presented as mean ± SEM from three independent experiments (n = 3). **P < 0.01; Student’s t-test. c Representative confocal laser scanning microscopy (CLSM) fluorescence (red) and visible chromogenic (brown) images of U87MG, SH-SY5Y, and CTX TNA2 cells after DGC labeling. d Representative CLSM images of U87MG cells labeled with DGC (red fluorescence) directly, or blocked with DAG peptide prior to DGC labeling (red fluorescence), or DGC (red fluorescence) co-staining with anti-CTGF antibody (green fluorescence). Data were from three independent experiments and one representative result is shown. Scale bar = 50 μm. Source data are provided as a Source Data file.
Fig 5: DGC multimodally analyzes CTGF in early AD brain sections of APP/PS1 mice in vitro.a Immunofluorescence staining of FITC-labeled CTGF antibody (green) and DGC (red) on APP/PS1 mouse brain sections and the age-matched wild-type (WT) brain sections from 1- to 9-month-old. The merge pictures show the colocalization of DGC and CTGF in brain sections. Scale bar = 50 μm. b In situ fluorescence imaging (red), Au elemental of LA-ICP-TOF-MS imaging, and 3, 3′-diaminobenzidine tetrahydrochloride (DAB) chromogenic (brown) imaging of CTGF in whole brain sections of WT mice (left), 3-month-old APP/PS1 mice (middle) and 9-month-old APP/PS1 mice (right). Inset pictures are enlarged areas of DAB chromogenic images. Scale bar = 50 μm. c CLSM fluorescence images of cortex region in brain sections of WT, 3-month-old APP/PS1 mice and 9-month-old APP/PS1 mice stained with DGC (red fluorescence) and FITC-labeled CTGF antibody (green fluorescence). Scale bar = 20 μm. Inset images show the colocalization of DGC and CTGF antibody. Scale bar = 10 μm. d CLSM fluorescence images of DGC (red fluorescence) and FITC-labeled-Aβ antibody (green fluorescence) staining show partial colocalization in cortex region in brain sections of WT, 3-month-old APP/PS1 mice and 9-month-old APP/PS1 mice. Scale bar = 50 μm. The inset pictures show the partial colocalization of DGC and Aβ antibody in 9-month-old APP/PS1 mice. Scale bar = 25 μm. Data were from three independent experiments.
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