Immunocytochemistry 6주차&7주차

Introduction Cytoskeleton Feature of cytoskeleton Maintains cell shape and assists movement of cell parts. Feature of cytoskeleton Dynamic structure Protect cell body. Enable cellular motion (flagella, cilia, lamellipodia) and intracellular transport (vesicles and organelles). Cellular division. The eukaryotic cytoskeleton Actin filament (microfilament). Microtubule. Intermediate filament.

Introduction Cytoskeleton inhibitors Blebbistatin Nocodazole Blebbistatin inhibits myosin ATPase activity and actomyosin based motility. Nocodazole Nocodazole is an antineoplastic agent which exerts its effect in cells by interfering with the polymerization of microtubules. Blebbistatin Nocodazole

Introduction Immunocytochemistry (ICC) Common lab practice which uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. In cases where an immune-positive signal is found, ICC allows researchers to determine which subcellular compartments are expressing the antigen. Monoclonal VS Polyclonal

Immunocytochemistry VS Immunohistochemistry

Introduction Procedures of immunocytochemistry Sampling Fixation Fixation is necessary to prevent artificial diffusion of soluble tissue component, to arrest enzymatic activity, to avoid decomposition of the structure, and to protect the tissue. All fixation protocol must ~ prevent antigen leakage. keep the antigen in such a form that it can be recognized efficiently by the antibody. maintain the cell structure. Ethanol, Methanol, Acetone, Formaldehyde, Paraformaldehyde, Glutaraldehyde

Introduction Blocking Permeabilization Blocking the reactive sites in tissues is essential for the development of an immunochemical reaction. A blocking solution is useful for reducing background staining. Blocking solution binds to any open protein binding sites that antibodies or antigens do not attach. Permeabilization The process of making something, such as a membrane or cell wall, permeable often through the use of surfactants. Permeabilization is necessary to allow access of the antibody. If the target protein is expressed intracellularly, it is very important to permeabilize the cells. If you used methanol or acetone to fix, permeabilization is not necessary. Triton X-100, NP-40, Tween 20 and Saponin.

Introduction Antibody binding Advantage Disadvantage Direct labeling of the primary antibody Indirect labeling of the primary antibody Advantage Cleaner signal. Weak background signal. Strong signal. Any reagent that will bind specifically to the primary antibody can be “tagged”. One set of labelled reagents can be used for a number of primary antibodies. Disadvantage The time is needed to purify and label each preparation of primary antibody. The signal will not be as strong as indirect methods. Background signal might be strong.

Introduction Washing Mounting Mounting provides the most permanent and secure means of long-term storage.

Introduction Reagent for immunocytochemistry Phalloidin DAPI Phalloidin binds to F-actin, preventing its depolymerization. Phalloidin labeled with a fluorescent dye is frequently used to visualize actin filaments by fluorescence microscopy. DAPI DAPI is a fluorescent stain that binds strongly to A-T rich regions in DNA.