SUBJECT

Immunocytochemistry

practical

master

Faculty of Science

Biology MSc

2

Semesters 1-4

Autumn/Spring semester

Theory:

Methods of protein separation from biological samples. Planning and cloning of an antigen of interest. Production of mono- and polyclonal antisera. Antisera characterization and antibody labelling.

Preparation of samples: collection, fixation and embeding of different biological samples.

Antigen retrieval technics and signal enhancement.

Bases of in situ hibridization and FISH methods.

Practice:

1. Immunohistochemistry Localization of different cytoskeletal proteins (cytokeratin, tubulin and actin) in the cells of fibroblast culture by using two-steps immunohistochemistry. Fluorescein microscopy and digital microphotography. Localization of a cuticular protein on paraplast-embedded sections from Manduca larvae by using enzyme -labelled second antibody. Light microscopical investigation and microphotography.

2. Electronmicroscopical immunocytochemistry Subcellular localization of heat-shock proteins in normal and pathological samples by using of colloidal gold labelled second antibody. Electronmicroscopical investigation and -photogaphy.

• Immunochemical Staining Methods Handbook. Ed. By Th. Boenish. 3rd edition, DAKO, 2001.

• David Wild: The Immunoassay Handbook, Newnes, 2013, ISBN 9780080970387