Fig. 4
Secondary antibody staining with multiple same-isotype primary antibodies and cell-surface molecule detection by ligand binding. (a) Summary of the steps to stain with an isotype-specific secondary antibody when its target isotype antibody is present multiple times in the same staining panel. (b) Staining of NKp46 with an isotype-specific secondary antibody (anti-mouse IgG1) in the presence of anti α4- and β1-integrin antibodies, which are of the same isotype as the anti NKp46 antibody. Peripheral blood mononuclear cells were pre-gated on single live lymphocytes as in Fig. 2a and CD3+ T cells and CD3−NKp46+ NK cells analyzed for expression of α4- and β1-integrins. (c) Corresponding isotype control staining for α4- and β1-integrins. (d) E-selectin ligand expression on CD4+ T cells from afferent lymph of adult sheep was determined by flow cytometry using an E-selectin-human IgG fusion protein. Cells were pre-gated as in Fig. 2a. As a negative control, staining was performed in buffer containing EDTA. (b-d) One representative of five individually analyzed sheep is shown. Abbreviations: αm, anti-mouse; APC, allophycocyanin; BUV, Brilliant ultra violet; FITC, fluorescein isothiocyanate; PE, R-phycoerythrin