Post-embedding immunocytochemistry for adhesive proteins and clathrin in LR White- and LR Gold-embedded human platelets

Abstract

Two hydrophilic acrylic resins, LR White and LR Gold, were tested for their suitability for post-embedding immunocytochemistry on human platelets. Both resins gave satisfying results with regard to ultrastructural preservation and retention of antigenicity for von Willebrand factor, fibrinogen, fibronectin and clathrin. Embedding in LR White and polymerization at 50 degrees C without accelerator proved to be the best method because it provided clear negative membrane contrast, good immunocytochemical localization of antigens and the entire procedure required a comparatively short time. Label for von Willebrand factor was found in typically eccentric location in alpha-granules, but also in the form of small caps directly outside the granule membranes, beneath the outer plasma membrane and in the cytoplasm. With the exception of the eccentric location within granules, labelling for fibrinogen and fibronectin revealed very similar results. Label for clathrin appeared in the form of small gold clusters attached to alpha-granules, in the cytoplasm and beneath membranes of the open canalicular system. Together with the appearance of coated vesicles attached to granules and to the membranes of the open canalicular system in araldite-embedded material, these results further strengthen the concept that adhesive proteins are taken up from the plasma and stored in granules. Moreover, the persistence of the clathrin coat on vesicles attached to storage granules may also indicate that receptors for adhesive proteins are recycled.