horse anti mouse secondary antibody conjugated to fluorescein  (Vector Laboratories)

Journal: Scientific Reports

Article Title: Construction of Vero cell-adapted rabies vaccine strain by five amino acid substitutions in HEP-Flury strain

doi: 10.1038/s41598-024-63337-9

Figure Lengend Snippet: Efficiency of viral growth, entry, and expression of G protein of each recombinant viruses in Vero cells. ( a ) Information on recombinant viruses used in this figure. *: Amino acid sequence identical with rHEP. **: Amino acid positions are numbered based on the mature G protein without signal peptide. ( b ) Vero cells were inoculated with the indicated recombinant viruses ( a ) at a M.O.I. of 0.05 and supernatants were collected every day until 4 d.p.i. Viral titers were determined in MNA cells. ( c ) The total number of focuses was determined by counting the number of focuses stained with the FITC anti-rabies monoclonal globulin in Vero or MNA cells under a fluorescence microscope. The ratio of the number of focuses in Vero to that in MNA was compared. ( d ) The particle titer of each secreted alkaline phosphatase (SEAP)-expressing VSVp stock was determined in MNA cells. Vero cells then were inoculated with two-fold serial dilutions (starting from 150 particles) of VSVp pseudotyped with the HEP or HEP-10V G protein SEAP activity was assessed in culture supernatants and detected by optical density (OD). ( e ) Vero cells were inoculated with each recombinant virus at an M.O.I. of 5 and harvested at 2 d.p.i. The cells were stained with anti-rabies G protein monoclonal antibody (#7-1-9) and FITC-conjugated anti-mouse secondary. After that, cells were fixed with 4% paraformaldehyde, and finally analyzed by BD FACS Canto II flow cytometer (Becton Dickinson and Company; BD, Franklin Lakes, NJ, USA) and Kaluza analysis software Version 2.1 (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Data are presented as the mean and S.D. from three ( b , d , e ) or four ( c ) independent experiments. Significant differences are indicated in the comparison between rHEP and each recombinant virus after application of two-way ANOVA followed by Tukey ( b ), or each virus after application of one-way ANOVA followed by Turkey ( c , e ) or two-way ANOVA followed by Sidaks ( d ) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Article Snippet: Cells were then washed twice and reacted for 1 h on ice with the FITC-conjugated Goat Anti-Mouse IgG1 secondary antibodies (1:1600) (FI-2000; Vector Laboratories, Newark, CA, USA).

Techniques: Expressing, Recombinant, Sequencing, Staining, Fluorescence, Microscopy, Activity Assay, Virus, Flow Cytometry, Software, Comparison