Derek Wong
Biology 275L-03
Lab 4
Immunofluorescence Microscopy of the Cytoskeleton Components
Introduction
Immunofluorescence (IF) is a technique used with antigen-antibody reactions in which antibodies are labeled by bonding a fluorescent dye to the immunofluorescence region of the antibody complex. Antibodies are part of the immune system which is the natural health defense system of the body. Whenever a foreign particle, or antigen, is present in the body, the immune system creates antibodies. These antibodies are produced by lymphocytes, specifically the B cell, help to control and eliminate antigens by binding to specific sites on the protein, called the antigenic determinant. When an antigen is bounded to an antibody, this creates an antigen-antibody reaction.
There are two main types of antibodies, polyclonal and monoclonal. Polyclonal antibodies are made with different specificity and can attack a multitude of antigenic sites on a protein. Unlike polyclonal antibodies, monoclonal antibodies (mAb) are produced specifically for one antigenic determinant. There are five classes of antibodies: IgG, IgE, IgD, IgA, and IgM.
In this experiment, a fluorochrome, specifically FITC and Texas Red will be conjugated to an antibody which will then bind to an antigen creating the antigen-antibody reaction. There are two ways to accomplish this: direct and indirect. Direct uses only one type of antibody conjugated with the fluorochrome to the antigen. Indirect uses two types of antibodies. The primary antibody will bind to the antigen and then the anti-antibody conjugated with the fluorochrome bind to that antibody. This experiment will be use the indirect method of labeling antigens, which will produce easier to see results because more secondary conjugated antibodies can bind to the antigen.
This experiment wills exam the cytoskeleton and its elements in the rat A-10 cells, specifically tublin, vimentin, and actin. Microtubules, responsible for cell motility and structural integrity, are the largest fibers (25nm) found in the cytoskeleton and are comprised of tublin. Microfilaments, responsible for cell mobility, are the smallest fibers (8nm) found in the cytoskeleton and are comprised of actin. Intermediate filaments (10nm), are in between the size of microfilaments and microtubules and are known for structural support.
Results
See next page for data and observations.
Discussion
In this experiment tublin, actin, and vimentin was examined in the rat A-10 cells using immunofluorescence microscopy with both the FITC and Texas Red filter.
Tublin is the unit of microtubles and under the immunofluorescence microscope, appeared to be red balls with the Texas Red Filter, and green ovals with red nucleus under FITC. Actin is the unit of microfilaments and appeared exactly as the tublin under FITC and under Texas Red. Vimentin helps make up intermediate filaments and under Texas Red appeared to be red string-like patterns stretching across the scope.
Immunofluorescence works by conjugating an antibody with a fluorochrome and then having that antibody attach to the antigen. When conjugating the antibody with the fluorochrome, it is important to add a blocking buffer. Blocking buffers help prevent the non-specific binding of the antibodies and to the cover slips and will cause the immunofluorescence not to work because the antigen-antibody complex will not be formed. The types of fluorochromes used were Texas Red and FITC. Although propidium iodide was used in this experiment to stain the nucleus so that it is easier to see, it is not a fluorochrome because it is not conjugated to an antibody.
For this experiment, the indirect method of labeling was used, meaning that both primary and secondary antibodies were used. It is important to note that the secondary antibodies must be complementary to the primary antibody (i.e. antibodies must come from the same species). For example, if you wanted to visualize vimentin in goat white blood cells using rat anti-goat as the primary antibody, the secondary antibody must be a type of anti-rat. In this example, the secondary antibody could not be a FITC labeled sheep anti-goat IgG because the anti-goat will not be able to conjugate to the primary rat anti-goat antibody.