HISTOLOGY BIOL-4000 LECTURE NOTES #2 MICROSCOPY AND HISTOCHEMISTRY CLASS POWERPOINT TEXT - DOWNLOADABLE POWERPOINT PRERSENTATION

MICROSCOPY
In examining slides of sectioned tissues with the light and electron microscopes, one should be aware that some of the structures observed may not be real, that is, they may be artifacts. Artifacts are the result of changes in a tissues structure or the addition of new structures that are not present in the living tissue and are usually the result of fixation, dehydration, embedding, sectioning, staining, and/or section mounting techniques. Types of artifacts that are commonly encountered are listed below. Light microscope examples of some of these are available for viewing in the digital lab manual available in the lab. REVIEW OF BASIC ARTIFACTS 1. Swelling of tissue components 2. Shrinkage of tissue components Artifact types 1 and 2 are the result of poor fixation and/or dehydration techniques, i.e. osmolarity of fixative may be wrong, pH wrong, too short a fixation time, dehydration of tissue too rapid. Swelling and shrinkage can sometimes result in rupture of membranes. This sort of damage is particularly evident at the ultrastructural level. 3. wrinkles in section 4. tears in section 5. air bubbles 6. dust Artifact types 3, 4, and 5 are usually the result of poor sectioning technique or poor technique during mounting of sections. In some cases, poor fixation and/or embedding can be responsible for tears or wrinkles in sections by modifying fixed tissue consistency such that the tissue cannot be sectioned without its tearing or wrinkling. 7. stain precipitate This sort of artifact can result from use of old stain solutions, use of unfiltered stain solutions, mistakes made during preparation of the stain, or poor staining technique. THE LIGHT MICROSCOPE. We have gone over the use of your light microscopes during lab and you there is a handout available on the class web site describing how to set-up your microscope for viewing such that "proper Kohler illumination" is established. In setting up "proper Kohler illumination" you are adjusting the path of the light such that a minimum of light reflection occurs within the microscope and the maximum amount of light passes through the various lenses within the microscope. In addition, light is restricted to the central portion of the lenses. The reason for this is that there are more optical defects in the image the lens produces as you move away from its center. The end result of your adjustments for "proper Kohler illumination" is that you achieve the maximum resolution of the image of the tissue you are viewing that is possible with your microscopes. This means that you will be able to see the maximum amount of structure within the tissue that can be seen with your microscopes. The objective and ocular lenses are responsible for magnifying the image of the specimen being viewed. Objective and ocular magnification are indicated as the number of times the image is magnified followed by the letter "X". Thus, a 10X objective or ocular will provide an image that is 10 times as large as the original tissue being viewed. Another way to say this is that if you are looking at an object that is 10 microns (um) long with a 10X objective and no ocular magnification, the image you will see will be 100 um long. When an objective lens and an ocular lens are used together (in series) to view an object magnification is calculated as follows: Total magnification = Objective magnification multiplied (x) by ocular magnification So for 10X objective and 10X ocular,

Total magnification = 10 x 10 = 100X (this means that the image being viewed will appear to be 100 times its actual size). For a 40X objective and 10X ocular, Total magnification = 10 x 40 = 400X Unfortunately, magnification, in itself, is not sufficient to look at the fine structure of tissues. It is also necessary that the resolving power of the lenses be high, i.e. resolution must be high as well as magnification. Resolution is a measure of the ability to distinguish 2 points as two points. That is, when viewing something through a microscope, how close together can two points be that you can still see some space between them? ** * * We can't say much more about resolution without a few words about numerical aperture (n.a. or NA). The value for numerical aperture measures to what extent the light that passes through a specimen is spread out over and collected by the objective lens. The light that passes through the specimen contains information about what the specimen looks like, that is, about its structure. If we consider the cone of light that originates from the specimen and enters the objective lens, Numerical aperture can be defined as, NA = n x sin a n = refractive index of substance between specimen and objective lens (usually air, n = 1.0; quartz, n = 1.5; glass, n= about 1.5; water, n = 1.3) a = 1/2 the aperture angle (also called the semiangle). The aperture angle is the angle described by the cone of light that enters the objective lens after passing through the specimen. This angle will depend on the curvature of the lens and also on how close the objective lens is to the specimen when it is in focus. So, for an objective with an aperture angle of 120 degrees with air between specimen and objective lens, NA = 1 X sin 60 degrees = sin 60 degrees = 0.87 If oil with refractive index of 1.5 is used between objective lens and specimen, NA = 1.5 X sin 60 degrees = 1.5 (.87) = 1.31 Numerical aperture is important because it allows us to calculate the resolving power of the objective. Remember, that's what we really were interested in determining initially. In general, the higher the numerical aperture, the better the resolving power of the objective. R = 0.61 X (lamda/NA) R = resolution of the objective lamda = wavelength of light (average value for white light (~550 nm). NA = numerical aperture So, for an objective with an NA of 0.87, R = 0.61 X 550nm/.87 = 386 nanometers (nm) = 386 X 10E-9 meters (m) = 0.000000386 meters (m) = 0.386 microns (um) For oil immersion using an objective with an NA of 1.31,, R = 0.61 X 550nm/1.31 = 256 nm = 0.000000256 m = 0.26 um Thus, one can see that higher resolution is possible if the substance lying between the specimen and the objective lens has a refractive index as close as possible to that of the lens itself without exceeding the lens' refractive index.It is important to realize that while both the ocular and objective lenses are responsible for the final magnification on a compound microscope, ONLY the objective lens and the substance lying between that lens and the object being viewed are responsible for resolution.

The discussion above should demonstrate the importance of resolution. By using the appropriate lenses I can create extremely high magnifications, say 5000X, with the light microscope. However, as stated above, magnification tells us nothing about resolution. If resolution of objective lens is 0.3 micron, no matter how much I magnify the specimen image, the resolution will remain the same. At 5000X, I will still only be able to resolve points a minimum of 0.3 micron apart. Points that are closer together may be visible, but they will be superimposed on each other and blurred, appearing as one fuzzy point. So nothing is gained by increased magnification if resolution is not also increased. Since the physics of light microscopy limits resolution for this type of microscopy to about 0.2 um, the amount of visible information available at 5000X is the same as at lower magnifications of 1500X (a magnification high enough to allow our eyes to see a resolution of 0.2 um). Using the mathematical equations given above and the values for maximum numerical aperture attainable with the lenses of a light microscope it can be shown that the maximum useful magnification on a compound light microscope is between 1000X and 1500X. Higher magnification is possible, but resolution will not improve. In addition to numerical aperture and an incorrect light path, there are 3 major lens defects that can affect the quality of the image in a compound microscope and result in decreased resolution. These are, A. Chromatic aberration - caused by spherical a lens bringing different wavelengths of light into focus at different levels. Thus, you get multiple images superimposed on top of each other. This defect is corrected in achromatic objectives. B. Spherical aberration - optical quality of image lessened due to the fact that the center of lens has slightly different qualities than the edges. Both spherical and chromatic aberration are corrected in apochromatic objectives. C. Curvature of field - causes image to be in focus centrally, but out of focus peripherally or vice versa. This defect is corrected in planar objectives. The type of objective, magnification, numerical aperture, and even the best coverslip thickness to use on your slides is listed on the side of an objective. You should know what resolution and numerical aperture are, in general, and the maximum resolution achievable on a standard compound light microscope, but you will not be expected to calculate these on an exam. LIGHT MICROSCOPY There are a number of special types of light microscopy that can enhance certain features of a specimen that is being examined. Some of these are listed below. 1. Phase contrast microscopy - takes advantage of phase differences in light beam that are caused by different refractive indexes of components within a tissue. Consider air, n = 1.0; water, n = 1.3; glass, n = 1.5. Light travels fastest through air and slowest through glass. Thus, if a light beam encounters three different spaces of equal thickness that are filled with air water, or glass, the beam will emerge first from the air filled space and last from the glass filled space. These light beams are said to be out of phase with each other. In the phase contrast microscope, the condenser and objectives are specially made to detect the phase differences of light passing through different components within a tissue specimen. The construction of the condenser and objective lenses is such that these phase differences are made visible by increasing the contrast between light waves of different phase. As a result, components of cells that are normally of low contrast (clear or nearly clear) are given higher contrast and caused to become visible. 2. Polarizing microscopy - A polarizing filter (called the polarizer) is placed below the condenser and allows light vibrating in only one plane to reach the condenser. A second polarizing filter (called the analyzer) is placed between the objective and ocular. If these two filters are oriented such that their axes of light transmission are perpendicular, no light will pass through the analyser to the ocular. So nothing will be seen. One use of polarizing light microscopy is related to the fact that certain crystals found in or associated with some cells can bend light waves because of their refractive index. If some of the light waves that initially passed through the polarizer are bent by crystals in a tissue into a different plane than the other light that passed through the polarizer, then some of these light waves

will be able to pass through the analyzer even though its axis of light transmission is oriented at 90 degrees to the axis of light transmission of the polarizer. This property of certain crystals to bend polarized light waves is called birefringence. The light that gets through the analyzer and is seen in such situations is called birefringence. Birefringence is important in identifying certain crystalline structures in or associated with cells. 3. Interference or Nemarski interference microscopy. - this is another method utilized to observe structures of different refractive index, but similar optical density. It is not the same as phase contrast microscopy. Nemarski interference microscopy requires two different light beams that are recombined after passing through the specimen. Differences in phase between the two beams are visualized as depth. The result is an image with depth (sort of 3-D). This type of microscopy is particularly useful for viewing living cells. It makes use of the difference in refractive index between the liquid component of the cell cytoplasm and the various organelles and other cytoplasmic structures. 4. Confocal microscopy - variable size apertures are used to eliminate out of focus light. This results in a very clear image and allows one to optically section through transparent and translucent specimens. In most instances a laser is used to scan the specimen and the light from the specimen is collected as a series of points (digital information) by a photomultiplier tube. Tissues are most often labeled with a specific fluorescent marker (e.g. an antibody conjugated to a fluorescent marker (fluor) such as fluorescene isothiocyanate - FITC) and the laser light of appropriate wavelength excites the fluor and causes it to fluoresce. This data is transferred to a computer where it is collected, analyzed and manipulated to produce very high resolution images. In some cases the data may be utilized to create 3-D reconstructions of the specimen. This type of microscope is a very power tool for research. ELECTRON MICROSCOPY Functioning of this instrument is dependent on the fact that an electron beam has many properties that are similar to a light beam. In fact, a beam of electrons may be treated as either 1.) a beam of particles or 2.) as a wave (i.e. like a light wave). As it turns out, both properties are necessary in order for an electron microscope to work. The fact that the effective wavelength of an electron beam is very much smaller than that of the shortest visible light wavelength makes very high resolution possible with this instrument (i.e. 5 - 20 angstroms) This means that very high useful magnification is possible since very small distances between two points can be resolved. The highest magnification commonly used with the electron microscope is 200,000X. However, higher useful magnifications are possible. Suffice it to say, that for the purposes of this course, we can consider the electron microscope in relatively simple terms. An electron beam is produced by inducing a high voltage between a cathode (-) and an anode (+). Electromagnets are used to direct that path of this beam and also to act as magnetic lenses that are responsible for magnification of the image of the specimen. As the electron beam passes through the specimen, electrons are either unaffected, scattered, or absorbed by the tissues of the specimen and various stains (usually heavy metals) that have been applied to the tissues. The unaffected electrons and many of the scattered electrons pass through the specimen and then are focused by magnetic lenses on a fluorescent viewing screen. The number of electrons hitting various parts of this screen determine how brightly these parts fluoresce and thus form an image of the specimen on the screen that can be examined by the person using the scope. In addition, the focused electrons can be used to expose photographic film from which black and white pictures can be printed. The photographs produced are actually more useful in interpreting electron microscope images because they are permanent and of higher contrast than the fluorescent image. HISTOCHEMISTRY BRIEF REVIEW OF FIXATION AND EMBEDDING A. Why fix tissue? 1. Preserve structure. Essentially to make the structural components of the tissue more durable. 2. Fixed material is dead. You want to preserve the structure (chemical and morphological) of the living material so that it appears the same as it was in life. 3. It will never be exactly the same. Important to choose fixative that does the best job. Fixative used will depend on type of tissue to be fixed. B. Why embed? 1. Tissue will be sectioned. Needs to be durable enough to withstand the sectioning process. Also, want components of tissue to remain in their natural positions. Don't want them to be moved to new positions.

2. Embedding in wax or plastic immobilizes structural components of tissue. Holds them in place as sectioning is done. C. Why section? 1. Allows you to see internal structure of tissue. 2. Allows stains, or specific markers such as antibodies to more easily infiltrate the tissues. 3. In some cases tissues are labeled without sectioning. D. Histochemistry: 1. Definition: Study of the chemical nature of cells and tissues with the light and electron microscopes. 2. Accomplished by using appropriate chemical analytical methods that result in visible changes in structure or color of components of the cells/tissues being examined. 3. These changes may be caused by the deposition of opaque or colored products from specific chemical reactions or they may be the result of stains or markers of some sort that bind or associate with specific chemical components of the cells/tissues. a. deposition of chemical reaction products, colored or opaque. b. stains or other types of specific substances that bind or associate with specific chemical components of the cells/tissues. 4. Histochemical reactions must meet 4 criteria in order to be considered a valid identification of some chemical component of a cell. a. Substance being analyzed must not diffuse out of its original site. b. Appropriate fixative, treatments, and embedding media must be used such that substance to be identified is not soluble in them. i.e. lipids - no non-polar solvents. c. reaction product must be colored, opaque or electron scattering so that it can be visualized. d. Method employed should be specific for substance being studied. If method is not totally specific, there must be controls that can be run that will eliminate other possible sites of reaction. e. Procedure must not block or inactivate reactive components being studied. 5. Other important factors a. Reaction product must be insoluble in the media used during the test so that it will not diffuse away from the original site where the substance being tested for was located. b. The histochemical test used must not destroy the structure of the tissue. EXAMPLES A. Deposition of chemical reaction products. 1. Suppose you wish to identify cells that catabolize hemoglobin such as certain cells in the liver. a. PERL REACTION - forms a reaction product with degraded hemoglobin b. Fixed tissue is treated with potassium ferrocyanide + HCl + Fe3+ = ferric ferrocyanide. Forms a dark blue precipitate when it reacts with degraded hemoglobin. B. Suppose you wish to study the role of Calcium salts (phosphates and carbonates) in bone histogenesis. 1. Von Kossa method (Mallory modification, 1944) a. Fix bone in 10% formalin or alcohol. b. Dehydrate, embed, and section c. Treat sections with 5% silver nitrate (AgNO3) for 30 min in dark d. Rinse sections in water e. Incubate sections in distilled water under bright light for1hr. f. Rinse thoroughly in distilled water g. Counterstain h. Dehydrate and mount 2. Calcium carbonate and phosphate deposits will be brown to black.

C. Locate polysaccharides in tissues 1. Polysaccharide - polymers of sugar molecules (i.e., chains of chemically linked sugar molecules, e.g., starch most common in plants, glycogen most common in animals.) 2. Glycogen - highly branched polymer of glucose (so it's a highly branched chain of glucose molecules). 3. Glycogen is used for energy storage in some animal cells, can be demonstrated by Periodic AcidSchiff reaction (PAS). a. Fix tissue - glycogen is water soluble, so tissue must be fixed in alcoholic (>70%) fixatives that will prevent the glycogen from being dissolved out of the tissues. b. Dehydrate, embed, section c. Deparaffinize and rehydrate sections d. Treat with periodic acid (HIO4), 5 min wash in water Treat with Schiff reagent (1% basic fuchsin + 1.9% sodium metabisulfite + 15% HCl in distilled water). 10 min e. Wash with 0.5% sodium metabisulfite followed by water and counterstain f. Dehydrate, clear, mount, and examine tissues on microscope. 4. Periodic acid converts the 1,2-glycol groups of the glucose molecules in the glycogen chain to aldehyde groups. 5. These aldehydes react with the bleached fuchsin dye in Schiff's reagent to form a new molecule having a purple color. So substances containing glycogen will appear purple when viewed with the light microscope. If the cells of a tissue have purple deposits after being treated with a PAS test, we say that they are PAS positive and that they contain stored polysaccharide in the region where the staining occurs. 6. However, some substances in cells that are not glycogen will also be PAS positive, so a proper control must be run. For animal tissue, one would treat some sections with an enzyme that degrades glycogen, such as salivary amylase and then treat slides with the PAS method. 7. Components of cells that are strongly purple when not treated with amylase, but lose their PAS reactivity when treated with amylase contain glycogen. a. PAS method + no amylase = intense purple color b. amylase treatment + PAS method = weak or no purple color 8. Further notes on the PAS method: a. Glycoproteins are usually identified by the fact that amylase treatment does not change their strongly PAS positive nature. b. Glycolipids may also be strongly PAS positive and not be affected by amylase. D. Other important histochemical treatments that result in the formation of colored or opaque deposits in tissues that identify the position of specific chemicals are those that have been developed for enzymes. You should look over examples in text for acid phosphatase, dehydrogenases, and peroxidase. 1. Peroxidase reaction a. Most common is reaction to identify peroxidase enzyme. This is because peroxidase is often linked to antibodies and the peroxidase reaction with a substrate and hydrogen peroxide is used to visualize where the antibody binds. b. Peroxidase - peroxidase will act as a catalyst causing a reaction between hydrogen peroxide and 3,3' diaminobenzidene (sometimes called "BAD DAB" because it is a strong carcinogen). The reaction product of this reaction is an insoluble, opaque, dark brown substance. 2. Specific stains that bind. RNA (ribonucleic acid) a. These molecules are basophilic - high affinity for basic stains b. They will stain intensely with the basic stains toluidene blue or methylene blue. c. Note that these stains will also bind to other cell components, so a control must be run. Pretreat some sections with ribonuclease, an enzyme that will degrade RNA. Then stain. Any structure that was

basophilic prior to ribonulcease treatment, but loses its basophlic character after treatment should contain RNA. 3. Association - stains that associate with a specific chemical component of a cell. a. Lipid specific stains - Sudan IV, Sudan Black. 1a. Fix tissue with 10% formalin 1b. Fixed tissue may be frozen and sectioned or used as a whole mount (unsectioned) if it's small enough. 1c. Dehydrate in a series of increasing concentration of ethylene glycol to 100% ethylene glycol. 1d. Transfer to solution of ethylene glycol saturated with Sudan IV for 1 - 10 hr depending on tissue. 1e. Rinse in 100% ethylene glycol to get rid of excess stain 1f. Mount tissue on slide in ethylene glycol and examine with microscope. 2a. Sudan IV has a high solubility in non-polar lipid. Non-polar lipid deposits in cells will stain red due to high concentrations of Sudan IV dissolved in them. 2b. For polar lipids, as well as non-polar lipids, Sudan Black is effective, Lipids stained black.

You will not be expected to know all the steps in the Histochemical procedures described above, but you should know what sort of substance they are specific for.
EXAMPLES OF HISTOCHEMICAL STAINING (**Under construction**) A. Reaction product deposition - OsO4 (Osmium tetroxide) lipid identification with light microscope Os04 binds to lipid. The reaction forms a black, opaque reaction product. 1. Wholemounts of fixed unstained larvae. Below, A. is a newly hatched veliger larva of the nudibranch sea slug, Tritonia diomedea. Note that there are no refractive droplets in the left digestive diverticulum (LDD) of the newly hatched larva. B. is a larva that has been feeding on algal plankton for about 30 days and that is nearly ready to undergo metamorphosis and form a juvenile slug. As you can see, the left digestive diverticulum is packed with large "clear" refractive droplets. These droplets might be lipid deposits that are stored as an energy reserve to provide energy for the juvenile slug after it metamorphoses. An osmium tetroxide reaction can be used to determine whether or not these droplets actually are lipid.

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2. Sections of the 30 day labeled larvae treated with OsO4 have circular, dark black areas of reaction product where the OsO4 reacted with the droplets that appeared refractive in fixed unstained larvae. This identifies the contents of those droplets as lipid.

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B. Association (in this case dissolution) of stain with specific chemical component of cell. 1. Sudan IV is a stain that is specific for non-polar lipids such as triacylglycerides. a. Wholemounts of fixed unstained larvae show round refractive droplets in the LDD as was the case with the 30 day old larva above.

b. In wholemounts of these larvae stained with Sudan IV the refractive droplets stain a bright red with Sudan IV, indicating that they are composed of non-polar lipid(s)
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2. Two or more of the above methods may be used in combination along with various types of microscopy. For example, combination of both the Sudan IV and OsO4 methods and light and electron microscopy to make specific identification of lipid deposits in muscle of a nudibranch larva such as the one above. Lipid droplets in retractor muscle of Melibe species. a. In the unstained larva it is difficult to see any indication of possible lipid droplets in the larval retractor muscle. b. Using the OsO4 reaction and using the electron microscope to observe apropriately prepared sections, a black reaction product indicating droplets of lipid can be see adjacent to mitochondria in the retractor muscle (retractor muscle is striated muscle). Why might the lipid droplets be next to the mitochondria?

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c. Sudan IV stained larva - using the light microscope red stained droplets of possible lipid are evident in retractor muscle. IMMUNOCYTOCHEMISTRY

A. Immunological techniques are becoming increasingly important in histology. These techniques are now being applied to nearly every facet of biological science, from systematics (taxonomy) to the investigation of carcinogenesis. They are also important diagnostic tools. B. If I had to pick any one facet of histological technique as being the most important or the potentially most useful technique, I would have to say that immunocytochemistry is it. C. This technique takes advantage of the fact that many animals have immune systems that will produce antibodies that react with a specific molecule (usually protein, but sometimes carbohydrate or lipid component of protein). This is true for nearly any type of proteineceous molecule that the cells of the immune system encounter and the antibody can be highly specific (i.e. the antibody will bind to no or only a few other molecules). Antibodies may also bind to peptides, carbohydrates associated with glycoproteins, lipids associated with lipoproteins, and even single amino acids if an appropriate antigenic substance is used to produce them. D. These antibodies can be used to identify and localize specific molecules within tissues, cells, or sub-cellular structures. This being the case, such antibodies can be used to look at the structure of specific organelles by virtue of the fact that some of their molecules will be unique within the cell, for instance, certain enzymes are found only within the mitochondrion. Or certain proteins are found only as structural units of microtubules, Or certain monoamines are used only as neurotransmitters/neurohormones (e.g. 5-hydroxytryptomine = 5-HT = serotonin). E. As I'm sure most of you are aware, antibodies can also be used to combat disease. Innoculations with inactivaed pathogens or inactivated toxins produced by a pathogenic organism can cause an immune response in you body that will enable your own immune system to produce antibodies against these pathogens or toxins, and thus protect you from disease. F. Antibodies that bind to specific proteins found only in certain types of cancer cells may be useful in treating certain kinds of cancers. One method involves linking a cell toxin or a radioisotope to the antibodies, injecting them into the person with the disease, and allowing the circulatory system to distribute them to the diseased tissues. The antibodies will then bind to the cancer cells and not to other cells in the body, thus concentrating the isotope or toxin in the tissues that you want destroyed. This would be one means of delivering high doses of cell toxin or radioactivity only to the cancer cells and not to other healthy cells of the body. G. In basic histological research, the high specificity possible with antibodies allows their use in identifying specific chemicals or structures within cells. H. The antibodies themselves do not allow us to visualize the cell components, rather, a marker such as a fluorescent compound, enzyme, or electron scattering particle is linked to an antibody. So where the antibody binds will be where this marker appears in the sectioned tissue. I. Two approaches to developing antibodies for histological uses. 1. Direct method - marker, such as a molecule that will fluoresce under a specific wavelength of light, conjugated (hooked) directly to an antibody that binds to a specific molecule within a tissue: a. Tissue is incubated in a solution of this marked antibody b. then rinsed to get rid of excess unbound antibody. c. Tissue is examined on microscope under illumination with the wavelength of light that will cause the fluor to fluoresce, thus revealing the location of the molecule of interest.

2. Indirect method: a. Tissue incubated with a solution of the primary antibody - this is the antibody that binds to the specific molecule of interest within a tissue. b. Tissue rinsed to remove excess unbound antibody. c. Tissue incubated with a secondary antibody - this is an antibody that will bind to the primary antibody. An example would be a goat secondary antibody that binds to mouse primary antibodies. The secondary antibody has a fluorescent molecule conjugated to it. The secondary antibody