Anti icp4 primary antibody

Indicated cell types were plated at 70%–80% confluency in twelve-well tissue-culture-treated dishes. The following day, cells were infected with a MOI of three. One and a half hours following infection, viral input was removed with a 20% glycine solution diluted in PBS. Six hours following infection, medium was removed and cells trypsinized. Cells were fixed with 4% paraformaldehyde (PFA) (Fisher Scientific) for 30 min at room temperature, then washed with PBS. Cells were then permeabilized with a 0.1% Triton X-100 (Sigma) solution for 10 min at room temperature and washed with PBS. Cells were then incubated in a 10% horse serum solution diluted in PBS for 1 h at room temperature. Following, cells were incubated with the anti-ICP4 primary antibody (Santa Cruz) overnight at 4°C. Cells were washed with PBS then incubated with the secondary anti-mouse Alexa 488 conjugated antibody (Fisher Scientific) for 1 h at room temperature in the dark. Cells were washed with PBS, and data were collected using a BD Accuri c6 Plus. Data were analyzed using FlowJo version 10.3.

Genome copy number was determined as previous described.³⁸ (link) Briefly, the entire infected tumor-bearing brain hemisphere was harvested. Total DNA was extracted using the QIAGEN DNA blood and tissue extraction kit. gc titers were calculated relative to a standard curve generated for each experiment using a 10-fold dilution series of plasmid pUL5 (corresponding to 3 × 10⁶ to 3 × 10² copies of the HSV genome) and a custom FAM-MGB TaqMan primer probe set (UL5qPCR-F, UL5qPCR-R, UL5 MGB probe; Thermo Fisher Scientific). In vivo immunofluorescence was performed by snap freezing infected tumor-bearing brain hemispheres in OTC (Tissuetek) freezing compound using liquid nitrogen. 12 μm sections were produced using a Cryostat Microm HM5050E. Permeabilized sections were stained for 48 h with anti-ICP4 primary antibody (Santa Cruz) and anti-nestin primary antibody (Santa Cruz). Alexa 488 and Alexa 594 conjugated secondary antibodies (Molecular Probes) were incubated for an hour at room temperature. DAPI staining took place for 10 min at room temperature. Images were captured using Nikon Diaphot fluorescence microscope (Nikon, Melville, NY, USA) and MetaMorph imaging software (Molecular Devices, San Jose, CA, USA).