Nitric Oxide Synthase Immunostaining

Mononuclear cells were separated from the bone marrow cells with lymphocyte separation medium (LSM, Capricorn Scientific, Ebsdorfergrund, Germany) according to the manufacturer’s instruction. For cytoplasmatic staining, mice bone marrow cells as well as CFU-E and BFU-E / CFU-GM colonies from methylcellulose cultures were collected onto microscope glass slides, as previously described [5 (link)]. Briefly, after fixation and incubation with the anti-neuronal NOS (nNOS, cat no. sc-5302 (1:100), Santa Cruz Biotechnology), anti-inducible NOS (iNOS, cat. no. sc-651 (1:100), Santa Cruz Biotechnology), and anti-eNOS antibodies (cat. no. sc-654 (1:100), Santa Cruz Biotechnology) immunostaining was performed using a computer-supported imaging system (analysis Pro 3.1) connected to the light microscope (Olympus AX70, Hamburg, Germany) with an objective magnification of ×40.