1. Immunocytochemistry
6주차&7주차

2. 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.

3. 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

4. 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

5. Immunocytochemistry VS Immunohistochemistry

6. 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

7. 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.

8. 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.

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

10. 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.