Description

Immunoelectron microscopy of mammary gland tissue from lactating rats with medial cisternae of the Golgi apparatus indicated by immunogold labeling of MG-160, a 160 kDa membrane sialoglycoprotein specific to medial Golgi.

Technical Details

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 incubation with an antibody to MG-160 (mouse monoclonal) followed by colloidal gold-conjugated secondary antibody (10 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. See Ladinsky and Howell (2007) for more information on methods used.