Schematic representation of intravital microscopy protocols. (A) In protocol 1, after anesthesia and surgical preparations, SCD mice were injected intraperitoneally (i.p.) with murine TNF-α (time 0) and then immediately with GMI-1070, or an antibody mixture containing anti–P- and –E-selectin monoclonal antibodies, or PBS through an intracarotid artery catheter (i.c.). To ensure activities of the selectin antagonist during the entire course of studies, SCD mice received a second dose of antagonists or vehicle controls 70 minutes after the first injection (T₇₀). Images of the cremasteric venules under intravital microscopy were recorded between the time points of 90 and 150 minutes (T₉₀ → T₁₅₀). During filming, the hemodynamic parameters, including centerline velocity, venular diameter, and shear rate, were measured. (B) Protocol 2 was designed to assess the therapeutic effects of GMI-1070 on SCD mice with ongoing acute veno-occlusive crisis primed by TNF-α. SCD mice were infused with GMI-1070 at 110 minutes after animals were challenged with TNF-α (time 0). Images were recorded in the 2-hour interval between 120 and 240 minutes (T₁₂₀ → T₂₄₀) after the administration of GMI-1070.