Journal: International Journal of Molecular Sciences

Article Title: Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain

doi: 10.3390/ijms21238951

Figure Lengend Snippet: Representative T2- and T2*-weighted images in sham (top three rows) and MCAO (bottom three rows) animals are shown for day 28 after injection of viral vectors. Immunofluorescent staining were done for ferritin (FerrH) and activated microglia/macrophages (CD68). eGFP signal was read at the fluorescent microscope with a wavelength of 488 nm. Areas of ischemic lesion were delineated on T2-weighted and propagated to T2*-weighted and to the histological images (bottom three rows). Green arrows point to areas of the transgene expression near the SVZ outside of the ischemic lesion . Abbreviations: MCAO, middle-cerebral-artery occlusion; AAV, adeno-associated viral backbone; pDCX, doublecortin promoter; FerrH, ferritin heavy chain; eGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; SVZ, subventricular zone.

Article Snippet: The immunocytochemistry staining was done using the following primary antibodies: goat anti-doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA, USA sc-8066, 1:100) to visualize immature neurons, rabbit anti-ferritin (Abcam, Cambridge, MA, USA, ab75973, 1:100) to visualize ferritin accumulation, rabbit anti-NeuN (MilliporeSigma, Burlington, MA, USA, ABN78, 1:200) to visualize mature neurons, and rabbit anti-CD68 (Wako Pure Chemical Industries, Richmond, VA, USA, 019-19741, 1:500) to visualize activated microglia.

Techniques: Injection, Staining, Microscopy, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain

doi: 10.3390/ijms21238951

Figure Lengend Snippet: Histological validation of the MRI signal hypointensity areas at day 28 after AAV-pDCX-FerrH, AAV-pDCX-eGFP, or PBS with fluorescent images of eGFP signal, FerrH-, and CD68-stained brain sections. Panels ( a – c ) show linear regression plots of the percentage changes in T2* signal hypointensity relative to the baseline point as in the ischemic lesion relative to the symmetric contralateral anatomical region as a function of ( a ) FerrH IF in the zone near the SVZ, ( b ) eGFP IF in the zone near the SVZ, and ( c ) FerrH IF in the ischemic lesion. Points represent color-coded individual data for the animals from different groups. Abbreviations: MCAO, middle-cerebral-artery occlusion; AAV, adeno-associated viral backbone; pDCX, doublecortin promoter; FerrH, ferritin heavy chain; eGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; SVZ, subventricular zone; red IF, intensity of red channel immunofluorescence in micrographs; green IF, intensity of green channel immunofluorescence in micrographs.

Article Snippet: The immunocytochemistry staining was done using the following primary antibodies: goat anti-doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA, USA sc-8066, 1:100) to visualize immature neurons, rabbit anti-ferritin (Abcam, Cambridge, MA, USA, ab75973, 1:100) to visualize ferritin accumulation, rabbit anti-NeuN (MilliporeSigma, Burlington, MA, USA, ABN78, 1:200) to visualize mature neurons, and rabbit anti-CD68 (Wako Pure Chemical Industries, Richmond, VA, USA, 019-19741, 1:500) to visualize activated microglia.

Techniques: Staining, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain

doi: 10.3390/ijms21238951

Figure Lengend Snippet: Examples of immunofluorescent staining for FerrH, NeuN (mature neurons), DCX (young neurons), and activated microglia\macrophages (CD68). ( a ) Double-immunofluorescence images stained for mature (FerrH\NeuN for the Ferr-MCAO and PBS-MCAO group, eGFP\NeuN for the eGFP-MCAO group), young (FerrH\DCX for the FerrH-MCAO and PBS-MCAO group, and eGFP\DCX for the eGFP-MCAO group) neurons in the zone near the lateral ventricle and SVZ. ( b ) Double-immunofluorescence images stained for microglia\macrophages (FerrH\CD68) in the near lateral ventricle and SVZ (left three rows) and inside the ischemic lesion (right three rows). For eGFP-MCAO group, FerrH is visualized by far-red fluorochrome and is shown as green pseudo color. Scale bars corresponds to 50 µm. Abbreviations: MCAO, middle-cerebral-artery occlusion; AAV, adeno-associated viral backbone; pDCX, doublecortin promoter; FerrH, ferritin heavy chain; eGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; SVZ, subventricular zone.

Article Snippet: The immunocytochemistry staining was done using the following primary antibodies: goat anti-doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA, USA sc-8066, 1:100) to visualize immature neurons, rabbit anti-ferritin (Abcam, Cambridge, MA, USA, ab75973, 1:100) to visualize ferritin accumulation, rabbit anti-NeuN (MilliporeSigma, Burlington, MA, USA, ABN78, 1:200) to visualize mature neurons, and rabbit anti-CD68 (Wako Pure Chemical Industries, Richmond, VA, USA, 019-19741, 1:500) to visualize activated microglia.

Techniques: Staining, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain

doi: 10.3390/ijms21238951

Figure Lengend Snippet: Cellular composition of two zones of MRI signal hypointensity in T2*-weighted images, near the SVZ and inside of the ischemic lesion area. ( a ) Percentage of mature (NeuN+) and immature (DCX+) neurons and activated microglia\macrophages (CD68+) in the zone near the lateral ventricle and SVZ in animals injected with AAV-pDCX-FerrH, ( b ) AAV-pDCX-eGFP, and ( c ) in the zone of ischemic lesion. Statistically-significant differences in percentage of cells according to ANOVA after LSD’s correction for multiple comparisons: in comparison with the percentage of NeuN+ cells,* p < 0.05 and *** p < 0.001; in comparison with the percentage of CD68+ cells, # p < 0.05 and ### p < 0.001. Abbreviations: MCAO, middle-cerebral-artery occlusion;, AV, adeno-associated viral backbone; pDCX, doublecortin promoter; FerrH, ferritin heavy chain; eGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; SVZ, subventricular zone.

Article Snippet: The immunocytochemistry staining was done using the following primary antibodies: goat anti-doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA, USA sc-8066, 1:100) to visualize immature neurons, rabbit anti-ferritin (Abcam, Cambridge, MA, USA, ab75973, 1:100) to visualize ferritin accumulation, rabbit anti-NeuN (MilliporeSigma, Burlington, MA, USA, ABN78, 1:200) to visualize mature neurons, and rabbit anti-CD68 (Wako Pure Chemical Industries, Richmond, VA, USA, 019-19741, 1:500) to visualize activated microglia.

Techniques: Injection

Journal:

Article Title: Infection and Injury of Neurons by West Nile Encephalitis Virus

doi: 10.1128/JVI.77.24.13203-13213.2003

Figure Lengend Snippet: WNV infection of differentiated ES cells. (A) Flow cytometric scatter and fluorescence dot plots of WNV-infected ESNC. Differentiation with retinoic acid yielded two populations of neurons by forward scatter (FSC) and side scatter (SSC) analysis (top). The larger population expressed higher levels of neurofilament (NF) and WNV antigens (compare middle and bottom panels) after infection. (B) Dose-dependent infection of ESNC. ESNC from 129/Sv (top) or C57BL/6 (bottom) mice were infected with increasing amounts of WNV and harvested 2 days later for flow cytometric and viral plaque assays. For flow cytometric analysis, neuronal populations were separated by their relative expression (low or high) of neurofilament protein. Data are the averages of three independent experiments and error bars reflect the standard deviations. (C) Kinetics of WNV infection in ESNC. Neurons were infected at an MOI of 0.05 with WNV. At the indicated times after infection, cells were harvested for flow cytometric analysis of viral antigen and supernatants were assessed for infectious virus as described for Fig. ​Fig.2.2. Data are the averages of three independent experiments and error bars reflect the standard deviations.

Article Snippet: After 4 days, EB were induced for neuronal differentiation by the addition of all- trans retinoic acid (Sigma Chemical) (500 nM) and were cultured again for 4 days, with medium changes.

Techniques: Infection, Fluorescence, Expressing