The α-Gal content is similar in humans and zebrafish. The α-Gal content was determined in zebrafish tissues and gut bacterial microbiota and in R. sanguineus salivary glands. (A) The α-Gal levels were determined by ELISA in zebrafish muscle, liver/kidney, and gut and in comparison with pork kidney (α-Gal positive) and human HL60 cells (α-Gal negative) as positive and negative controls, respectively. The results were converted to α-Gal content per sample using a calibration curve (R2 = 0.992; Supplementary Figure 5A) and compared between all samples and negative (lines) or positive (*p < 1E-8) controls by Student t-test with unequal variance (p < 0.05, N = 5 biological replicates). (B) Flow cytometry showing the presence of α-Gal on the surface of aerobic and anaerobic bacteria isolated from zebrafish gut microbiota. Escherichia coli O86:B7 and BL21 (DE3) strains were included as positive and negative controls for α-Gal, respectively. For flow cytometry, cells were stained with Bandeiraea simplicifolia I-isolectin B4–FITC to visualize α-Gal, and the viable cell population was gated according to forward-scatter and side-scatter parameters. (C) The MFI was determined by flow cytometry, and the geometric mean ± SD compared between aerobic and anaerobic bacteria by Student t-test with unequal variance (p = 0.05, N = 5 biological replicates). (D) Distribution of the MFI among aerobic and anaerobic type bacteria in wild-type AB and pet store zebrafish and in comparison with E. coli O86:B7 and BL21 (DE3)–positive and –negative controls for α-Gal, respectively. The results (average ± SD) were compared between all samples and negative control by Student t-test with unequal variance (*p < 0.05, N = 5 biological replicates).
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