Fig. 2

NSG-cmah^−/− mice phenotype. a Western blot analysis of Neu5Gc presence in NSG wild type mice (cmah⁺) and NSG-cmah^−/− (cmah-) tissue samples. All tested mouse tissues with chicken anti-Neu5Gc antibody (Biolegend, CA, USA, 1:4000) were negative. b Confirmation of CMAH knockout on NSG background by immunohistology. Spleen, kidney, and lung tissues were fixed, paraffin embedded and 5 μ thick sections of NSG-Cmah^−/− generated strain (left), wt NSG (right) mouse and Cmah^−/-C57/Bl6 original immune competent strain (middle column) were stained with antibodies for Neu5Gc (anti-Neu5Gc antibody, Biolegend, CA, USA, 1:100) at 4 °C overnight. Images captured by Nikon E800 microscope at objective magnification 20×. New generated strain-derived tissues deficient for Neu5Gc as existing cmah^−/-C57/Bl6 strain. Wt NSG mice express Neu5Gc epitopes, and tissue sections have brown color. c Confirmation of CMAH gene knockout on NSG background by FACS. We compared expression of Neu5Gc on white blood cells by staining with anti-Neu5Gc antibody and secondary FITC-labeled anti-chicken reagent. Panel shows Neu5Gc staining for C57Bl/6 cmah^−/− original strain obtained from the Jackson Laboratories (Stock No: 017588) (red) compare to C57Bl/6 wild type (orange). d Panel shows the similar pattern of the absence of Neu5Gc expression on NSG-cmah^−/− mice (cyan) and the presence of Neu5Gc on cells derived from heterozygous mice that retain enzyme activity and have Neu5Gc on the surface of leucocytes (green and red). c, d Individual samples of a mean fluorescence intensity (left panels) and mean ± SEM are shown for each group of mice (n = 6, right panels)