Author/Editor		Zavašnik-Bergant, Tina; Bergant, Martina; Jeras, Matjaž; Griffiths, Gareth
Title		Preparation of cryo sections and quantitative immunogold electron microscopy - a case study on protease inhibitor in immune cells
Type		članek
Source		In: Gajić S, editor. Proceedings 2nd Croatian congress on microscopy with international participation. Zagreb: Croatian society for electron microscopy,
Publication year		2006
Volume		str. 102-3
Language		eng
Abstract		Summary. Endogenous protease inhibitor (cystatin C) localization was determined by quantitative immunogold electron microscopy, performed on labelled cryo sections of human dendritic cells. Differentiation of monocytes and maturation of immature dendritic cells were applied as an in vitro model systems for studying transport pathway of this endogenous inhibitor in immune cells. Introduction. Dendritic cells play a crucial role in efficient antigen-specific immune response. Their maturation occurs as they migrate from peripheral tissues, where they search for antigen, to lymphoid organs, where they present captured antigen to specific T cell receptors. Limited antigen degradation, performed by proteolytic enzymes (cathepsins) inside endocytic pathway and their potential control with intracellular protease inhibitor (cystatin), is a crucial step in generating antigenic peptides which can be recognized by specific T cell receptors. In presented study dendritic cells were used as a cell model in which a quantitative electron microscopy (1) was applied for studying endogenous and added cystatin C. Protease cathepsin S was used as a potential target enzyme as well as an organelle marker for MHC II loading compartments. Materials and methods. Immature dendritic cells were generated from isolated blood monocytes and further stimulated with tumour necrosis factor-alpha or lipopolysaccharide. Cells were fixed with 4% paraformaledhyde, embedded into gelatin, infused with sucrose and cut to ultrathin cryo sections (60 - 70 nm) at -120°C. Cryo sections were labelled with specific rabbit antihuman cystatin C and rabbit anti-human cathepsin S polyclonal antibodies and Protein A-gold (10 nm). Sections were contrasted with uranyl acetate and viewed with Philips CM120 Biotwin transmission electron microscope. (Abstract trunacted at 2000 characters)
Descriptors		DENDRITIC CELLS MICROSCOPY, ELECTRON CYSTATINS IMMUNOHISTOCHEMISTRY