Hematek 3000 system

Peripheral blood cells from E9.5 to E12.5 embryos were collected as detailed above and cytospun onto glass slides at 400 g for 10 min (Cellspin^® III, Tharmac). Slides were stained with Modified Wright’s Stain on a Siemens hematek 3000 system. The slides were viewed using an Olympus BX52 microscope (Olympus, Tokyo, Japan) with an Olympus UPlanApo 100x/1.35 NA Oil Iris objective (1,000 × total magnification). Micrographs were taken with an Olympus DP72 digital camera and with CellSens image acquisition software (Olympus, Tokyo, Japan).

All tests were performed manually, including packed cell volume (PCV), red blood cell (RBC) count, white blood cell (WBC) count, WBC differential (e.g., neutrophil, eosinophil, heterophil, monocyte, and lymphocyte), thrombocyte count, total plasma protein concentration, and hemoglobulin concentration. PCV was obtained by hematocrit tubes. Blood cells were counted in a Neubauer hemocytometer. The blood films were stained with Wright–Giemsa (Siemens Hematek 3000 System), and the white blood cell differential was read at 100× oil objective. Cells were identified according to published images (Arnold, 2005 (link)). Total plasma protein concentration was determined with a refractometer (Lines and Raine, 1970 (link)). Hemoglobin quantitation was performed using the sodium lauryl sulfate method (Karsan et al., 1993 (link)). Three shark individuals in each immunization program were involved in the above analyses.