Goat anti rabbit igg dylight 594

Hela cells were seeded onto glass coverslips and after 24 hrs were infected at a MOI of 1. At 24 hrs post infection, cells were fixed with methanol and blocked using 1% BSA in TBS-TX (25mM Tris-HCl, pH7.5; 150mM NaCl; 0.1% Triton X-100). Cells were stained with appropriate primary antibodies which were observed by staining with goat anti-rabbit IgG-DyLight 594 and goat anti mouse IgG-DyLight488 secondary antibodies (Jackson Immunoresearch). Images were captured on a Nikon Eclipse 80i fluorescence microscope and analyzed using Nikon Elements software.

MA104 cells were transfected with 100 nmol miR-7 mimic, inhibitor and negative oligonucleotide for 24h before RV infection. Then, the cells were infected with rotavirus at a multiplicity of infection (MOI) of 0.5. Cells were assayed for infectious rotavirus by fluorescent assay using rabbit anti rotaviral VP7 antibody (Genscript, Nanjing, China) at 24 hpi. Cells were fixed with 2% paraformaldehyde containing 0.2% Triton X-100 for 30 min at 4 °C, then fixed with pre-cooled 98% methanol for 10 min at 4 °C. After washing with ice-cold PBS, samples were blocked with 2% bovine serum albumin (Sigma) in PBS for 1 h at 37 °C. The slides containing cells were incubated with antibodies, such as rabbit anti-VP7 (0.1 μg/mL, Genscript, Nanjing, China) and rabbit anti- NSP5 (0.1 µg/mL, Genscript, Nanjing, China) for 1.5 h at 37 °C. After incubation, cells were washed with PBS and incubated with goat anti-rabbit IgG-DyLight™ 594 (0.5 μg/mL, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at 37 °C. The slides were then washed three times with PBS and covered with a cover slip, followed by staining with 4, 6-diamidino-2- phenylindole dihydrochloride (Sigma, USA). The negative controls were performed in parallel. Samples were analyzed using a fluorescence microscope (Nikon, Tokyo, Japan). The average numbers of positive cells in at least 5 fields were quantified in different group.