Journal: Virology

Article Title: The OC43 human coronavirus envelope protein is critical for infectious virus production and propagation in neuronal cells and is a determinant of neurovirulence and CNS pathology

doi: 10.1016/j.virol.2017.12.023

Figure Lengend Snippet: HCoV-OC43 lacking E protein can infect neuronal cells but replication is severely impaired. LA-N-5 (A) and mixed primary mouse CNS cells (B) were infected with supernatant coming from BHK21 supernatant (P0) and containing virus lacking E protein, rOC/E-Stop + pcDNA(empty), or initially complemented virus, rOC/E-Stop + pcDNA(OC-E). Cell-free and cell-associated virus infectious titers were determined over 72 h post-infection. Representative of three different experiments. Statistical significance was tested at 72hpi (*** P < 0.001). (C) IFA on infected mixed primary murine CNS cells over 48 h. Green represents the microtubule associated protein 2 (MAP2) staining in neurons; red represents viral S protein.

Article Snippet: For primary mouse CNS cultures, after blocking with a PBS-BSA 2% (wt/vol) solution for one hour at room temperature, primary antibody polyclonal rabbit anti-S protein (dilution 1/1000) and mouse monoclonal antibody against the neuron-specific MAP2 protein (1/1000; BD Pharmagen, catalog no. 556320) were diluted in PBS + 0.1% Triton X-100 and incubated on cells for one hour at room temperature followed by three PBS washes.

Techniques: Infection, Virus, Staining

Journal:

Article Title: In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

doi: 10.1128/JVI.77.18.9799-9808.2003

Figure Lengend Snippet: Gene transduction into primary rat cerebellar cells with AcVSVG-CAGFP. Primary rat cerebellar and hippocampal cultures were infected with AcVSVG-CAGFP. Immunofluorescence was examined by confocal microscopy. (A, D, and G) Anti-GFP immunochemistry. (B) Anti-Calbindin immunochemistry as a purkinje marker. (E) Anti-GFAP immunochemistry as a glial marker. (H) Anti-MAP2 immunochemistry as a neuronal marker. C, F, and I are merged images.

Article Snippet: Cells were then double labeled with either rabbit anti-GFP (Molecular Probes, Eugene, Oreg.), mouse anti-Calbindin (Swant, Bellinzona, Switzerland), mouse anti-MAP2 (ICN Biomedicals, Costa Mesa, Calif.), or mouse anti- glial fibrillary acidic protein (GFAP) (Zymed Laboratories, Inc., South San Francisco, Calif.) for 30 min to 1 h at 37°C.

Techniques: Transduction, Infection, Immunofluorescence, Confocal Microscopy, Marker

Journal:

Article Title: In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

doi: 10.1128/JVI.77.18.9799-9808.2003

Figure Lengend Snippet: GFP expression in mouse brains after cerebral injections of AcVSVG-CAGFP. Mice were injected with 4 × 107 PFU of AcVSVG-CAGFP in the right lateral ventricle. GFP expression in the brain was examined by fluorescent stereomicroscopy 2 days after injection. (A) Panels A to D are stereomicroscopic images of whole brain (A and B) and brain cross sections (C and D). Panels A and C are bright-field views, while panels B and D are fluorescent views. Arrows and dark staining indicate the injection route, as the infiltrated viral inoculum contained 0.04% trypan blue. (B) Immunohistochemical staining of the cryostat sections was examined by fluorescence microscopy following staining with antibodies specific for GFP (A and D), GFAP as a glial marker (B), or MAP2 as a neuronal marker (E). Panels C and F are merged images.

Article Snippet: Cells were then double labeled with either rabbit anti-GFP (Molecular Probes, Eugene, Oreg.), mouse anti-Calbindin (Swant, Bellinzona, Switzerland), mouse anti-MAP2 (ICN Biomedicals, Costa Mesa, Calif.), or mouse anti- glial fibrillary acidic protein (GFAP) (Zymed Laboratories, Inc., South San Francisco, Calif.) for 30 min to 1 h at 37°C.

Techniques: Expressing, Injection, Staining, Immunohistochemical staining, Fluorescence, Microscopy, Marker