Immunoelectron microscopy of mammary gland tissue from lactating rats investigating potential structural interactions between mature lipid droplets and elements of the trans-Golgi network, identified by TGN38 (a transmembrane trans-Golgi protein; larger gold particles) and cation-dependent mannose-6-phosphate receptor (an integral membrane protein that localizes to the trans-Golgi reticulum, endosomes, and the plasma membrane; smaller gold particles) staining. Note that trans‐Golgi cisternae in lactating mammary cells contain large amounts of casein and other milk proteins and are grossly expanded due to the osmolarity of the secreted fluid.
Tissue was processed for immunoelectron microscopy using a modified Tokuyasu method. Briefly, minced tissue was fixed in 4% paraformaldehyde containing 5% sucrose and 100 mM HEPES and infiltrated with PBS containing 2.1 M sucrose over 10 h, with repeated solution changes. Fixed tissue was transferred to an aluminum cryosectioning stub (Ted Pella, Inc., Redding, CA) and immediately frozen in liquid nitrogen. Semithin (90 nm) cryosections were cut at -110C with an UltraCut UCT/FCS cryomicrotome (Leica), using a diamond knife (Diatome) and transferred to a Formvar-coated, carbon-coated, glow-discharged 100-mesh copper-rhodium electron microscopy grid. Following blocking of nonspecific antibody binding sites with 10% calf serum in PBS, the sections were labeled by sequential incubation with antibodies to cation-dependent mannose-6-phosphate receptor (rabbit) and TGN38 (mouse monoclonal 2F7) and colloidal gold-conjugated secondary antibodies (10 nm anti-rabbit, 15 nm anti-mouse; Ted Pella Inc., Redding, CA) and then negatively stained and embedded with 1% uranyl acetate, 1% methylcellulose in distilled water. Samples were viewed in a Philips CM10 electron microscope, and images were collected digitally. Magnification 15,500 X. See Ladinsky and Howell (2007) for more information on methods used.
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