Cell Biology 2021 Part I_.pdf

Cell Biology 1 Prof. Bashar Saad CELL BIOLOGY PREPARED BY PROFESSOR BASHAR SAAD ARAB AMERICAN UNIVERSITY JENIN 2020/21 Cell Biology 2...

Cell Biology 1 Prof. Bashar Saad CELL BIOLOGY PREPARED BY PROFESSOR BASHAR SAAD ARAB AMERICAN UNIVERSITY JENIN 2020/21 Cell Biology 2 Prof. Bashar Saad Text Books: 1. Molecular Biology of the Cell Sixth Edition By: Bruce Alberts, Dennis Bray et al., Published by Garland Publishing ISBN-13: 978-0815344322 2. Biology Tenth Edition. By Campbell. ISBN-13: 978-0321775658 Cell Biology 3 Prof. Bashar Saad 1. Introduction............................................................................................................5 1.1 Milestones in Cell Biology...............................................................................5 1.2 The cell............................................................................................................7 1.3 Characteristics of living things.........................................................................9 2. How we study cells............................................................................................... 11 2.1 Microscopy....................................................................................................... 11 2.1.1 The light microscope:.................................................................................. 13 2.1.1.1 Bright field microscopy:........................................................................... 16 2.1.1.2 Dark Field Viewing.................................................................................. 19 2.1.1.3. Phase Contrast Microscopy..................................................................... 20 2.1.1.4 Microscopy with Oil Immersion............................................................... 21 2.1.2 Using a Counting Chamber......................................................................... 23 2.1.3 Fluorescence Microscopy............................................................................... 25 2.1.4 Confocal Microscopy..................................................................................... 26 2.2 Electron Microscopy.......................................................................................... 27 2.3 Cell Fractionation............................................................................................... 30 2.4 Immunocytochemistry....................................................................................... 32 2.5 Cell & Tissue Culture......................................................................................... 32 2.6 Antibodies Can Be Used to Detect and Isolate Specific Molecules.................. 33 3. Internal Organization of the Cell......................... Error! Bookmark not defined. 3.1 Plasma membrane structure and function........... Error! Bookmark not defined. 3.1.1 Membrane Structure..................................... Error! Bookmark not defined. A. Introduction:.................................................. Error! Bookmark not defined. B. The Lipid Bilayer.......................................... Error! Bookmark not defined. C. Membrane Proteins........................................ Error! Bookmark not defined. D. Membrane Carbohydrate............................... Error! Bookmark not defined. 4. CELL COMMUNICATION (CHAPTER 11)............... Error! Bookmark not defined. 4.1 Introduction:.................................................... Error! Bookmark not defined. 4.2 How simple organisms communicate ?............ Error! Bookmark not defined. 4.3 How cells of multicellular organisms communicate ?..... Error! Bookmark not defined. 4.4 Cellular communication is divided into three steps......... Error! Bookmark not defined. 4.4.1 Reception..................................................... Error! Bookmark not defined. 4.4.2. Signal-transduction pathways:..................... Error! Bookmark not defined. 4.4.3 Cellular response:......................................... Error! Bookmark not defined. 6. The organization and control of eukaryotic genomes......... Error! Bookmark not defined. 6.1 Introduction:....................................................... Error! Bookmark not defined. 6.2 The structure of chromatin:............................. Error! Bookmark not defined. 6.3 Genome organisation at the DNA level........... Error! Bookmark not defined. 6.4 Gene organisation:.......................................... Error! Bookmark not defined. 6.5 Gene amplification, loss, and rearrangement... Error! Bookmark not defined. 6.6 The control of gene expression........................ Error! Bookmark not defined. Cell Biology 4 Prof. Bashar Saad 6.7 The molecular biology of cancer..................... Error! Bookmark not defined. 6.7.1 Cancer cells have escaped from the cell-cycle control systems:............Error! Bookmark not defined. 6.7.2. Cancer results from genetic changes that affect the cell cycle:.............Error! Bookmark not defined. 6.7.3. Multiple mutations underlie the development of cancer:... Error! Bookmark not defined. 7. Animal development.............................................. Error! Bookmark not defined. (Chapters 46 and 47)................................................... Error! Bookmark not defined. A. Introduction.................................................... Error! Bookmark not defined. B. Mammalian development................................ Error! Bookmark not defined. 1. Spermatogenesis:.............................................. Error! Bookmark not defined. 2. Oogenesis:......................................................... Error! Bookmark not defined. 3. Fertilisation and early development:.................. Error! Bookmark not defined. 4. The stages of early embryonic development...... Error! Bookmark not defined. a. Fertilisation:...................................................... Error! Bookmark not defined. b. The main functions of fertilisation..................... Error! Bookmark not defined. c. The acrosomal reaction:..................................... Error! Bookmark not defined. d. Membrane depolarisation:................................. Error! Bookmark not defined. e. The cortical reaction:......................................... Error! Bookmark not defined. f. Activation of the egg:......................................... Error! Bookmark not defined. g. Cell cleavage:.................................................... Error! Bookmark not defined. 8. The genetic basis of development............................ Error! Bookmark not defined. From single cell to multicellular organisms........... Error! Bookmark not defined. Embryonic development:...................................... Error! Bookmark not defined. The study of development:.................................... Error! Bookmark not defined. Differential gene expression.................................. Error! Bookmark not defined. Genetic and cellular mechanisms of pattern formation......... Error! Bookmark not defined. Cell Biology 5 Prof. Bashar Saad 1. INTRODUCTION Cell Biology  Is the study of structure and function of cells and their organelles.  One of the main subjects of cell biology is the study of the relationship between structure and function.  Cell biology deals also with regulation of cell proliferation, cell differentiation, cell-to-cell interactions, and cell to substrate interactions. 1.1 Milestones in Cell Biology 1626 Redi postulated that living things do not arise from spontaneous generation. 1655 Hooke described 'cells' in cork. 1674 Leeuwenhoek discovered protozoa. He saw bacteria some 9 years later. 1833 Brown descibed the cell nucleus in cells of the orchid. 1855 Virchow postulated that new cells come from preexisting cells. 1857 Kolliker described mitochondria. 1869 Miescher isolated DNA for the first time. 1879 Flemming described chromosome behavior during mitosis. 1883 Germ cells are haploid, chromosome theory of heredity. 1898 Golgi described the golgi apparatus. 1926 Svedberg developed the first analytical ultracentrifuge. 1938 Behrens used differential centrifugation to separate nuclei from cytoplasm. 1939 Siemens produced the first commercial transmission electron microscope. 1941 Coons used fluorescent labelled antibodies to detect cellular antigens. 1952 Gey and co-workers established a continuous human cell line. 1953 Crick, Wilkins and Watson proposed structure of DNA double-helix. 1955 Eagle systematically defined the nutritional needs of animal cells in culture. Meselson, Stahl and Vinograd developed density gradient centrifugation in 1957 cesium chloride solutions for separating nucleic acids. Ham introduced a defined serum-free medium. Cambridge Instruments 1965 produced the first commercial scanning electron microscope. Cell Biology 6 Prof. Bashar Saad Sato and collegues publish papers showing that different cell lines require 1976 different mixtures of hormones and growth factors in serum-free media. production of monoclonal antibodies 1978 The production of monoclonal antibodies. Cell Biology 7 Prof. Bashar Saad The cell  Cells are the basic unit of structure and function of organisms:  All living organisms are made from cells  Your body has many different kinds of cells. Average number 1013.  Most cells have chemical and structural features in common.  In humans, there are about 200 different types of cells  Cells contain about 20 different types of organelles.  Cells are always created and destroyed in the human body:  About 300 million cells are produced every minute in our bodies  (3 x 109 nucleotides/cell x 300 million = 9 x 1017 nucleotides produced each minute)  About 300 million cells die every minute in our bodies!  Cells are separated from the environment by a plasma membrane.  (109 lipid molecules in the plasma membrane x 300 million = 3 x 1015 lipid molecules produced each minute)  Cells derive only from cells  Structurally we can differ between two cell types: 1. Prokaryotic cells (bacteria) 2. Eukaryotic cells (all other cells)  There are about 10 times as many microbial cells in the human body as there are human cells; around 1014 bacterial cells  They live in symbiosis Cell Biology 8 Prof. Bashar Saad Prokaryotic cells vs Eukaryotic cells: Prokaryotic cells Eukaryotic cells No nuclear membrane Definite membrane bound nucleus No membrane bound organelles Contains membrane bound organelles Size 0.1m - 10m. 10m-100m. Their DNA is located in the nucleoid. DNA in the nucleus Most of them have a cell wall With or without a cell wall Have smaller ribosomes with different Have typical ribosomes RNA and proteins than eukaryotic cells Cell Biology 9 Prof. Bashar Saad Characteristics of living things Living things have a variety of common characteristics. 1. Organisation. Living things exhibit a high level of organization, with multicellular organisms being subdivided into cells, and cells into organelles, and organelles into molecules, etc. 2. Homeostasis.  Homeostasis is the maintenance of a constant (yet also dynamic) internal environment in terms of temperature, pH, and water concentrations, etc.  Much of our own metabolic energy goes toward keeping within our own homeostatic limits.  If you run a high fever for long enough, the increased temperature will damage certain organs and impair your proper functioning.  Muscular activity generates heat as a waste product. This heat is removed from our bodies by sweating. Some of this heat is used by warm-blooded animals, mammals and birds, to maintain their internal temperatures. 3. Adaptation.  Living things are suited to their mode of existence.  Charles Darwin began the recognition of the marvellous adaptations all life has that allow those organisms to exist in their environment. 4. Reproduction and heredity.  Since all cells come from existing cells, they must have some way of reproducing, whether that involves asexual or sexual.  Most living things use the chemical DNA as the physical carrier of inheritance and the genetic information. Cell Biology 10 Prof. Bashar Saad  Some organisms, such as retroviruses, such as HIV use RNA as the carrier. The variation that Darwin and Wallace recognised as the wellspring of evolution and adaptation, is greatly increased by sexual reproduction. 5. Growth and development.  Even single-celled organisms grow.  When first formed by cell division, they are small, and must grow and develop into mature cells.  Multicellular organisms pass through a more complicated process of differentiation and organogenesis (because they have so many more cells to develop). 6. Metabolism  Living things exhibit a rapid turnover of chemical materials, which is referred to as metabolism.  Metabolism involves exchanges of chemical matter with the external environment and extensive transformations of organic matter within the cells of a living organism.  Metabolism generally involves the release or use of chemical energy. Nonliving things do not display metabolism 7. Detection and response to stimuli (both internal and external). 8. Interactions.  Living things interact with their environment as well as each other.  Organisms obtain raw materials and energy from the environment or another organism.  The various types of symbioses (organisms interact with each other) are examples of this. Cell Biology 11 Prof. Bashar Saad 2. HOW WE STUDY CELLS  Studying cells helps us understand how organisms’ function.  Cellular components work together to carry out life functions.  Cellular processes enable organisms to meet their basic needs.  The discovery and study of cells was dependent on the development of the microscope.  Further refinements and adaptations have allowed study of subcellular components at the molecular level. Here, we review some of the common methods used to study cells and tissues. 2.1 Microscopy There are two types of microscopes: 1. the light microscope 2. the electron microscope Cell Biology 12 Prof. Bashar Saad Microscope Light microscope Electron microscope Visible Light Fluorescence microscope microscope SEM TEM Bright field Confocal Phase Fluorescence contrast microscope Dark field Cell Biology 13 Prof. Bashar Saad The light microscope:  The light microscope, so called because it employs visible light to detect small objects, is probably the most well known and well-used research tool in biology.  Under the standard light microscope thin sections of tissues are examined by transillumination. Light is passed through a condenser that collects the light into a focused beam that then passes through the specimen and is collected by an objective lens that magnifies the image (4X to 100X).  The specimen is viewed through an ocular lens that also magnifies the image (usually 10-15X). The total magnification is obtained by multiplying the magnifying power of the objective and ocular lenses. Cell Biology 14 Prof. Bashar Saad  Image quality is dependent on the resolving power of the microscope, which is largely determined by the quality of the objective lens.  The light microscope has a limiting resolution (Resolution refers to the clarity of the specimen viewed under the scope ) of about 0.1 micron (objects smaller than this cannot be resolved) which allows a magnification of over 1000X without a loss of quality.  Most cells and tissues in their natural state contain little pigment that would absorb light. Therefore, they are normally transparent and little detail can be seen.  Consequently, methods have been developed to stain cells, subcellular components and tissue components and structures.  Most classical stains require a fixed or preserved specimen. Preservation of the specimen is necessary to preserve cellular structures in a "natural" state against intracellular digestion and desiccation.  When tissues or cells must be thinly sectioned, the aqueous environment is replaced by a resin or embedding media such as paraffin or epoxy that penetrates the cell and adds rigidity to the tissue.  These embedding media are usually not hydrophilic and sometimes lipids are removed.  To limit the loss of lipids or study enzyme activity, sometimes the tissues are cut when frozen rather than fixing and embedding them. In the ideal tissue preparation, the tissue retains the same structure and molecular composition as the living tissue. Cell Biology 15 Prof. Bashar Saad Types of light microscopes Bright field microscopy Dark field viewing Phase contrast Oil immersion microscopy Differential interference contrast Confocal microscopy Fluorescence microscopy Cell Biology 16 Prof. Bashar Saad 2.1.1.1 Bright field microscopy: A. Principles:  The bright field microscope is best known to students  Visible light is focused through a specimen by a condenser lens, then is passed through two more lenses placed at both ends of a light-tight tube. The latter two lenses each magnify the image.  Limitations to what can be seen in bright field microscopy are not so much related to magnification as they are to resolution and contrast.  Resolution can be improved using oil immersion lenses, and lighting and contrast can be dramatically improved using modifications such as dark field, phase contrast, and differential interference contrast. Fluorescence and confocal microscopes are specialized instruments, used for research, clinical, and industrial applications. Cell Biology 17 Prof. Bashar Saad B. When to use bright field microscopy:  Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms.  It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections.  Here is a not-so-complete list of specimens that might be observed using bright- field microscopy, and appropriate magnifications (preferred final magnifications are emphasised). -Prepared slides, stained - bacteria (1000x), thick tissue sections (100x, 400x), thin sections with condensed chromosomes or specially stained organelles (1000x), large protists or metazoans (100x). -Smears, stained - blood (400x, 1000x). - Algae and other microscopic plant material (40x, 100x, 400x).. Cell Biology 18 Prof. Bashar Saad C. Stereo microscope:  Other than the compound microscope, a simpler instrument for low magnification use may also be found in the laboratory  This is the stereo microscope, or dissecting microscope. Stereo microscopes usually have a binocular eyepiece tube a long working distance, and a range of magnifications, typically from 5x to 35 or 40x.  Cell Biology 19 Prof. Bashar Saad 2.1.1.2 Dark Field Viewing A. Principle  To view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye. Instead of coming up through the specimen, the light is reflected by particles on the slide.  Everything is visible regardless of color, usually bright white against a dark background. Pigmented objects are often seen in "false colors," that is, the reflected light is of a color different than the color of the object. B. When to use dark field viewing  Dark field illumination is most readily set up at low magnifications (up to 100x), although it can be used with any dry objective lens.  Dark field is especially useful for finding cells in suspension. Dark field makes it easy to obtain the correct focal plane at low magnification for small, low contrast specimens. Cell Biology 20 Prof. Bashar Saad 2.1.1.3. Phase Contrast Microscopy A. Principle  Most of the detail of living cells is undetectable in bright field microscopy because there is too little contrast between structures with similar transparency and no color.  However, the various organelles show wide variation in refractive index (the tendency of the materials to bend light) providing an opportunity to distinguish them.  Highly refractive structures bend light to a much greater angle than do structures of low refractive index.  The same properties that cause the light to bend also delay the passage of light by a quarter of a wavelength or so. In a light microscope in bright field mode, light from highly refractive structures bends farther away from the center of the lens than light from less refractive structures and arrives about a quarter of a wavelength out of phase.  Light from most objects passes through the center of the lens as well as to the periphery. Now if the light from an object to the edges of the objective lens is retarded a half wavelength and the light to the center is not retarded at all, then the light rays are out of phase by a half wavelength. They cancel each other when the objective lens brings the image into focus. A reduction in brightness of the object is observed. The degree of reduction in brightness depends on the refractive index of the object. Cell Biology 21 Prof. Bashar Saad Applications for phase contrast microscopy  Phase contrast is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed and the specimen is colorless or the details so fine that color does not show up well.  Cilia and flagella, for example, are nearly invisible in bright field but show up in sharp contrast in phase contrast. Amoebae look like vague outlines in bright field, but show a great deal of detail in phase.  Most living microscopic organisms are much more obvious in phase contrast. 2.1.1.4 Microscopy with Oil Immersion A. Principle  When light passes from a material of one refractive index to material of another, as from glass to air or from air to glass, it bends. Light of different wavelength bends at different angles, so that as objects are magnified the images become less and less distinct.  This loss of resolution becomes very apparent at magnifications of above 400x or so. In fact, as you will see later, even at 400x the images of very small objects are badly distorted.  Placing a drop of oil with the same refractive index as glass between the cover slip and objective lens eliminates two refractive surfaces and considerably enhances resolution, so that magnifications of 1000x or greater can be achieved. Cell Biology 22 Prof. Bashar Saad B when to use oil immersion lenses  Use an oil immersion lens when you have a fixed (dead - not moving) specimen that is no thicker than a few micrometers. Even then, use it only when the structures you wish to view are quite small - one or two micrometers in dimension.  Oil immersion is essential for viewing individual bacteria or details of the striations of skeletal muscle. It is nearly impossible to view living, motile protists at a magnification of 1000x, except for the very smallest and slowest.  A disadvantage of oil immersion viewing is that the oil must stay in contact, and oil is viscous. A wet mount must be very secure to use oil. Oil immersion lenses are used only with oil, and oil can't be used with dry lenses, such as your 400x lens.  Lenses of high magnification must be brought very close to the specimen to focus and the focal plane is very shallow, so focusing can be difficult. Oil distorts images seen with dry lenses, so once you place oil on a slide it must be cleaned off thoroughly before using the high dry lens again. Oil on non-oil lenses will distort viewing and possibly damage the coatings. Cell Biology 23 Prof. Bashar Saad 2.1.2 Using a Counting Chamber  For microbiology, cell culture, and many applications that require use of suspensions of cells, it is necessary to determine cell concentration.  One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types.  A device used for cell counting is called a counting chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts.  One entire grid on standard hemocytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares. Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus, the entire counting grid lies under a volume of 0.9 mm-cubed. Cell Biology 24 Prof. Bashar Saad  To get the final count in cells/ml, you to count the average number of cells per square, which has a volume of 0.1 mm3. Then multiply the result by the total surface area (104), (As cm3 = 10 mm3 and 1cm3 = 1ml). Sometimes you will need to dilute a cell suspension to get the cell density low enough for counting. In that case you will need to multiply your final count by the dilution factor. For example, suppose that for counting we had to dilute a suspension of hepatocytes 10 fold. Suppose we obtained a final count of 50 cells/square. Then the total cell number is suspension is 50 x 104 x 10 = 50 x 105 cells/ml. Cell Biology 25 Prof. Bashar Saad 2.1.3 Fluorescence Microscopy Fluorescence Many dyes exhibit the phenomenon of absorption when they are exposed to light. There are also compounds that emit part of the absorbed energy as light rays. This emission is known as fluorescence if the emission takes place very shortly after the absorption of light. Fluorescence Microscopy:  Utilizes substances (fluorophores) that, when irradiated by a certain wavelength of light emit light of a longer wavelength.  Tissue sections are usually irradiated with ultraviolet light so that the emission is in the visible portion of the spectrum. The microscope used has a strong ultraviolet light source and special filters that eliminate ultraviolet light before it gets to the observer’s eyes.  Some fluorescent compounds have an affinity for nucleic acids allowing the localization of nucleic acids with cells. Others are specific for the mitochondria. Fluorescent probes can be made by chemically coupling the fluorophore to a relevant molecule such as a hormone or antibody. Natural fluorophores include, the aromatic amino acids, NADH, flavins, and chlorophyll. Organic dyes (e.g., fluorescein, rhodamine), biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin). Cell Biology 26 Prof. Bashar Saad  Cell Biology 27 Prof. Bashar Saad 2.1.4 Confocal Microscopy  Confocal microscopy uses lasers and computers to produce three-dimensional images of living cells and tissue slices.  Optical sections of the specimen are collected by laser scanning and stored in the computer. The information from each visual plane can be viewed and reconstructed into three dimensional projections of the specimen  Confocal microscopy images light from a thin confocal slice rather than from the entire specimen.  Fluorescence from the specimen is focused by the objective lens through a pinhole aperture to a photomultiplier.  Another advantage is that it allows the optical sectioning of a three-dimensional object and subsequent three-dimensional visualization using 3-D rendering software. Cell Biology 28 Prof. Bashar Saad 2.2 Electron Microscopy  Electron Microscopy is based on the interaction of electrons with tissue components and electron dense stains.  The electron microscope is capable of 0.1 nanometer resolution. Magnifications can be as much as 400 times greater than those achieved in light microscopy. There are two types of electron microscopy: A. Transmission electron microscopy or TEM:  The principle behind transmission electron microscopy is that an electron beam is passed through a stained ultrathin section of a tissue that has been embedded in plastic. The thin sections are cut with diamond or glass knives and mounted on metal grids prior to staining.  The electron dense stain (lead or uranium) is differentially taken up by the cellular structures and therefore the electrons are absorbed more by some structures than others. The electrons that pass through the specimen fall on a photographic film or digital image plate to form an image.   Cell Biology 29 Prof. Bashar Saad B. Scanning electron microscopy (SEM):  Scanning electron microscope permits pseudo-three-dimensional views of the surface of specimens.  Specimens are covered with an electron dense material (such as gold, platinum, silver, and chromium) and a narrow electron beam in passed over the surface. At each point, the electron beam produces emitted electrons from the metal coating of the specimen. A detector captures these emitted electrons and the brightness of a synchronous cathode ray tube is modified to produce a three dimensional image that has highlights and shadows. Cell Biology 30 Prof. Bashar Saad 2.3 Cell Fractionation Cell Fractionation: This process of taking a cell apart, separating the major organelles so that their functions can be studied.  Separation of cellular components from one another is accomplished by differential centrifugation.  Centrifugal force is used to separate organelles and cellular components as a function of their sedimentation coefficients. The coefficient is based on the size, form, and density of the particle.  Cells are mechanically lysed and the homogenate is subjected to successive centrifugation at increasing speeds. Cell Biology 31 Prof. Bashar Saad  At each step the resultant pellet contains a different cellular component and the supernatant is collected for the next step.  For instance, the first step might pellet the nuclei leaving the other cellular components in the supernatant. After the second step mitochondria and lysosomes are collected in the pellet and the supernatant subjected to the next spin.  The size and density of the cellular components in the pellet decreases with each step in the process.  Cell fractionation allows detailed study of cellular components obtained in a relatively pure state. Cell Biology 32 Prof. Bashar Saad 2.4 Cell & Tissue Culture  Cell and tissue culture allow for the direct study of cell behavior in vitro. Chemically defined media, growth factors, hormones and serum components simulate the normal environment in a culture dish.  Infectious agents such as protozoa and viruses that grow only within cells can be studied using cell culture techniques. Cells are collected by enzymatic or mechanical disruption of tissues and isolated to culture media as suspension or contact dependent cultures.  Normal cells have finite, genetically programmed life span. However, transformed cells such as those found in cancer, become immortal cell lines and can go through many more cell divisions and have a much longer life.  Cells can be harvested and frozen in liquid nitrogen for later reconstitution and use in cell culture experiments. 2.5 Immunocytochemistry  Immunocytochemical methods allow the study of the presence and activity of specific macromolecules in cells and tissues.  Antigen-antibody, and receptor-hormone interactions are exploited by the labelling with fluorescent molecules, enzymes or electron dense molecules.  These labels make the molecule visible in the microscope without causing a loss of the protein's biologic activity.  Labelled antibodies or hormones bind only to their antigens or receptors, respectively, thereby permitting localization of specific antigens in tissue specimens.  In direct immunocytochemistry, an antibody binds to a specific antigen.  The more sensitive indirect approach uses an unlabeled primary antibody to the antigen and a labelled secondary antibody that binds to the primary antibody and makes the complex visible under the microscope. Cell Biology 33 Prof. Bashar Saad ***Indirect detection is more suitable for studies of poorly expressed antigens, which benefit from the signal amplification provided by the secondary reagent. 2.6 Antibodies Can Be Used to Detect and Isolate Specific Molecules  Antibodies are proteins produced by the vertebrate immune system as a defense against infection.  They are unique among proteins because they are made in billions of different forms, each with a different binding site that recognizes a specific target molecule (or antigen).  The precise antigen specificity of antibodies makes them powerful tools for the cell biologist.  Labelled with fluorescent dyes, they are invaluable for locating specific molecules in cells by fluorescence microscopy; labelled with electron-dense particles such as colloidal gold spheres, they are used for similar purposes in the electron microscope.  As biochemical tools, they are used to detect and quantify molecules in cell extracts and to identify specific proteins.  When coupled to an inert matrix to produce an affinity column, antibodies can be used either to purify a specific molecule from a crude cell extract or, if the molecule is on the cell surface, to pick out specific types of living cells from a heterogeneous population. Cell Biology 34 Prof. Bashar Saad Polyclonal antibodies  Polyclonal antibodies are made most simply by injecting a sample of the antigen several times into an animal such as a rabbit or a goat and then collecting the antibody-rich serum. This antiserum contains a heterogeneous mixture of antibodies (Polyclonal antibodies), each produced by a different antibody- secreting cell (B lymphocyte).  The different antibodies recognize various parts of the antigen molecule as well as impurities in the antigen preparation. Cell Biology 35 Prof. Bashar Saad Monoclonal Antibodies In 1976 the problem of antiserum heterogeneity was overcome by the development of a technique that revolutionized the use of antibodies as tools in cell biology.  The principle is to propagate a clone of cells from a single antibody-secreting B lymphocyte so that a homogeneous preparation of antibodies can be obtained in large quantities.  The practical problem, however, is that B lymphocytes normally have a limited life-span in culture.  To overcome this limitation, individual antibody-producing B lymphocytes from an immunized mouse or rat are fused with cells derived from an "immortal" B lymphocyte tumor.  From the resulting heterogeneous mixture of hybrid cells, those hybrids that have both the ability to make a particular antibody and the ability to multiply indefinitely in culture are selected.  These hybridomas are propagated as individual clones, each of which provides a permanent and stable source of a single type of monoclonal antibody.  This antibody will recognize a single type of antigenic site - for example, a particular cluster of five or six amino acid side chains on the surface of a protein.  Their uniform specificity makes monoclonal antibodies much more useful for most purposes than conventional antisera, which usually contain a mixture of antibodies that recognize a variety of different antigenic sites on even a small macromolecule.  But the most important advantage of the hybridoma technique is that monoclonal antibodies can be made against molecules that constitute only a minor component of a complex mixture. In an ordinary antiserum made against such a mixture, the proportion of antibody molecules that recognize the minor component would be too small to be useful.  But if the B lymphocytes that produce the various components of this antiserum are made into hybridomas, it becomes possible to screen individual hybridoma clones from the large mixture to select one that produces the desired type of monoclonal Cell Biology 36 Prof. Bashar Saad antibody and to propagate the selected hybridoma indefinitely so as to produce that antibody in unlimited quantities.  In principle, therefore, a monoclonal antibody can be made against any protein in a biological sample. Once the antibody is made, it can be used as a specific probe - both to track down and localize the protein that induced its formation and to purify the protein in order to study its structure and function. Since fewer than 5% of the estimated 10,000 proteins in a typical mammalian cell have thus far been isolated, many monoclonal antibodies made against impure protein mixtures in fractionated cell extracts identify new proteins.  Using monoclonal antibodies and gene-cloning technology, it is no longer difficult to identify and characterize novel proteins and genes. The problem is to determine their function, and the most powerful way of doing this is often by the use of recombinant DNA technology.