Sites of Glycosylation Observed by Postembedding and Preembedding Lectin Staining .

Abstract

Sites of glycosylation were investigated using postembedding and preembedding lectin staining. Positive labeling in the luminal side of perinuclear spaces and endoplasmic reticulum was observed by Nacetylgalactosamine and mannose binding lectins. Cis lamellae of the Golgi apparatus were stained by Nacetylgalactosamine and mannose binding lectins. Medial to trans lamellae were mainly labeled by galactose binding lectin and transmost lamellae were labeled by sialic acid and fucose binding lectins. The present results elucidated the route of glycoconjugate synthesis in relation to cell organelle.The Golgi apparatus is a polarized cell organelle that is involved in many important biological phenomena in the cell. This organelle is also regarded as a main center of glycosylation. To detect sites of glycosylation, many histochemical approaches have been tried (1, 3-8, 10, 11). In this presentation, postembedding and preembedding lectin staining methods were used to investigate sites of glycosylation. This approach is based on the principle of determining sites of glycosylation by detecting the initial appearance of sugar residues according to the route of synthesis of protein or glycoprotein, using lectins. MATERIALS AND METHODS Animals and Tissue PreparationDigestive tracts and other tissues were obtained from adult male Wistar rats under ether anesthesia. Specimens were cut into blocks and fixed overnight at 4°C in half strength Karnovsky's fixative. Then the tissue blocks were divided into two groups; one group was stained by the postembedding lectin staining method, and the other was stained by the preembedding lectin staining method. Postembedding lectin stainingAfter fixation, specimens were dehydrated through a graded ethanol series at progressively lower temperatures down to -35°C, and embedded in Lowicryl K4M. Ultraviolet polymerization of the resin was performed at -35°C for 24 hr, and subsequently at 20°C for an additional 24 hr, with a lowtemperature polymerization apparatus: TTP 010 (Balzers Union, Liechtenstein). Ultrathin sections were cut with a Reichert Ultracut N ultratome and mounted on nickel or gold grids precoated with Formvar film. The sections were rinsed for 20 min with PBS and then incubated for 60 min at room temperature in lectin-colloidal gold conjugate at a concentration 20-100 pg/ml. In some cases, 40 gum thick vibratome sections were first incubated in the incubation media of NADPase and TPPase. Ultrathin sections were cut from thick sections after polymerization and lectin colloidal gold conjugate were applied. As a control, 0.5 M haptenic sugar was preincubated with colloidal gold-labeled lectins for 1 hr at room temperature before application to the sections. The sections were washed with PBS and water and counterstained with uranyl acetate and lead acetate before electron microscopy. Preembedding lectin stainingFixed specimens were infused with a graded series

Glycosylation and sorting pathways of lysosomal enzymes in mussel digestive cells

“…Pre-embedding lectin cytochemistry Small pieces of tissue were fixed in diluted Karnovsky at 4°C overnight (Murata et al 1994), cryoprotected with sucrose and frozen by immersion in liquid nitrogen. Cryosections (10 μm) were obtained with the aid of a Leica CM300 cryotome under constant temperature (−30°C), treated with phosphate buffer (PBS) plus 1% BSA (PBS-BSA) for 10 min and stained overnight with HRP-labelled or DIG-labelled lectins diluted in PBS-BSA (Table 3).…”