The Cd41 antibody binds cd41:EGFP-positive cells in zebrafish peripheral blood with >99% sensitivity and specificity and is specific for Cd41. (A) The Cd41 antibody recognizes cd41:EGFP-positive cells in zebrafish peripheral blood with >99% sensitivity and specificity by flow cytometry. (B) Light scatter characteristics demonstrate that double-positive cells (black) are mostly located within the “lymphoid” gate (red dashed line) when overlaid on live cells (blue). Contour levels are set at 10% and outliers are shown. The “precursor” gate is also shown for reference (purple dashed line). (C) Wright-Giemsa stain of cytopreparation showing that Cd41 antibody-positive cells are generally thrombocytes based on morphology. (D) Flow cytometry of zebrafish peripheral blood reveals a loss of Cd41 antibody staining in cd41 mutant zebrafish. There is no reduction in the percentage of antibody-stained Cd41-positive cells in heterozygous zebrafish. (E) The median fluorescence intensity of cells from heterozygous zebrafish is about two-thirds that of wild-type (WT) zebrafish. (F) In spleen cells from adult zebrafish, cd61b:EGFP is expressed by a subset of Cd41 antibody-positive cells, but populations of both cd61b:EGFP single-positive and Cd41 antibody single-positive cells also exist. By light scatter characteristics, the cd61b:EGFP single-positive cells (green) map to a slightly different location than the overlapping double-positive cells (black) and Cd41 antibody single-positive cells (purple). Live cells are shown in the background (blue). Contour levels are set at 10% and outliers are shown. APC-A, allophycocyanin-area; FSC-A, forward scatter-area; SSC-A, side scatter-area.