Refrigeration up-regulates glycosidases to the surface of platelets. (A-C) Glycosidase localization in platelets. Immunofluorescence micrographs of fixed and permeabilized resting platelets with the use of anti-sialidase 1 (Neu1) or anti–β-galactosidase (β-Gal) Abs and Alexa-conjugated secondary Abs (green or red). Note the granular pattern of Neu1 and β-Gal staining in the permeabilized platelets (panels A and B, respectively). (C) The combined fluorescence is shown, and some, but not all, of the vesicular structures costain. (D-F) Glycosidase localization in refrigerated platelets. Nonpermeabilized (No Perm) refrigerated (4°C, 48 hours) human platelets were labeled with the anti-Neu1 (D) anti–β-Gal Abs (F). (E) Residual staining for Neu1 in platelets shown by detergent permeabilization (Perm). Scale bar, 5 μm. (G) Sialidase activity associated with fresh (room temperature, RT) or refrigerated human platelets (4°C, 48 or 96 hours). Total sialidase activity in platelets was measured in permeabilized room temperature platelets (n = 3); *P < .05. The amount of 4-methylumbellyferone released in quenched reaction mixtures was measured in 96-well Microfluor-1 Black plates on a Spectra MAX GEMINI EM Microplate Spectrofluorometer (Molecular Devices) with excitation, emission, and cutoff wavelengths of 355, 460, and 455 nm, respectively. Background fluorescence was subtracted from each data point.