Tissue Preparation, Microscopy & Electrophoresis PDF

# Classification of The Microscopes According to Light Source ## I- Microscopes using visible light source: 1. Optical light microscope. 2. Modified microscopes 3. Phase contrast microscope, which is used in the field of tissue culture. 4. Interference microscope. 5. Polarizing microscope. 6. Dark...

# Classification of The Microscopes According to Light Source ## I- Microscopes using visible light source: 1. Optical light microscope. 2. Modified microscopes 3. Phase contrast microscope, which is used in the field of tissue culture. 4. Interference microscope. 5. Polarizing microscope. 6. Dark field microscope 7. Dissecting microscope (Stereomicroscope), which is used in the field of surgery. ## II- Microscopes using invisible beam: 1. Ultraviolet microscope. 2. X-ray microscope. 3. Electron microscope, which may be of two types: * **A**. Transmission electron microscope. * **B**. Scanning electron microscope. # Tissue Preparation ## 1. Tissue sampling A small piece of tissue (biopsy) is obtained under anaesthesia or taken immediately after death (autopsy). ## 2. Fixation * **Fixation** is the treatment of the tissue with chemical or physical agents to avoid tissue autolysis (Digestion by enzymes present within the cells). A small piece of organ removed from the body should be treated by fixatives as soon as possible. Examples of fixatives are formaldehyde and glutaraldehyde. * After fixation, the sample is dehydrated in a series of increasing alcohol concentrations to prevent rapid shrinkage of the tissue sample. * 50 % Alcohol * 70 % Alcohol * 90 % Alcohol * 100 % Alcohol ## 3. Embedding * **Embedding** is the process of making the sample more solid so that it can be sliced. This is usually done by infiltrating the sample with a substance such as paraffin wax or resin. ## 4. Sectioning * Once the tissue is embedded, it is sliced into thin sections using a **microtome**. * The *microtome* is a machine that is equipped with a sharp steel blade which cuts the tissue into very thin slices. * The thickness of each of these slices ranges between 3-5 µm. * These sections are then mounted on glass slides. * **Freezing technique** is an alternative to the paraffin embedding and sectioning technique, in which the tissue is frozen using liquid nitrogen, and thin sections (3-5 µm) are cut inside a cold cabinet using a machine called a **cryostat**. ## 5. Staining * The sections are then stained with dyes to make the different components of the tissue visible. * **Hematoxylin** is a basic dye that colors the acidic components of the cell with a bluish tint. Examples of acidic components are the nucleus (DNA, RNA) and regions of the cytoplasm rich in ribosomes. * These components that are stained with hematoxylin are referred to as **basophilic**. * **Eosin** is an acid that stains the basic components of the cell a pinkish color. For example, most of the cytoplasmic components have a basic pH, so they are stained pink. * The cytoplasmic elements that are stained with eosin are said to be **acidophilic**. | Reagent | Result | |--------------|---------------------------------------------------------------------------------------------------| | Hematoxylin | Blue: nucleus, acidic regions of the cytoplasm, cartilage matrix | | Eosin | Pink: basic regions of the cytoplasm, collagen fibers | ## 6. Mounting * Once the tissue section is stained, it is mounted on a glass slide, which is then ready for observation under a microscope. # Protein Electrophoresis - A laboratory technique in molecular biology for separating molecules (DNA, RNA, proteins) based on their charge and/or size. ## 1. Sample preparation * Many molecules (amino acids, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them. * The sum of these charges determines the overall charge. At neutral pH: * Proteins have a unique electrical charge. * Both DNA and RNA bases are negatively charged. ## 2. Electrophoresis * The sample is loaded into a gel, which is a porous material made of **polyacrylamide**. * Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix suitable for the separation of proteins of typical size. * **Sodium dodecyl sulfate (SDS)** is a detergent that denatures proteins and gives them a negative charge. * An electric current is then applied to the gel, which causes the negatively charged molecules to migrate through the gel. * The smaller molecules will migrate faster than the larger molecules. ## 3. Visualization * **Ethidium bromide** is a dye that is used to visualize DNA. * When exposed to UV light, ethidium bromide fluoresces, allowing visualization of DNA. * **Bromphenol blue** is a dye used to visualize the sample (tracking dye). * **Glycerol** is added to the sample to increase its density which helps to make the sample fall to the bottom of the well rather than floating up. * **Staining** is done overnight with agitation, which facilitates penetration and helps ensure uniformity of staining. # Prokaryotes vs Eukaryotes ## Prokaryotes - Prokaryotic cells are the most primitive cells. They do not have a definite nucleus. - DNA floats around in the cytoplasm, protected by restriction enzymes. * Restriction enzymes cut foreign DNA (invading viruses) into small pieces. * DNA is not cut by these restriction enzymes as there is a methyl group attached to DNA. ## Eukaryotes - Eukaryotes contain a definite nucleus. - All eukaryotes have DNase enzymes that can cut DNA. # Cell Lysis - The process of breaking open cells to release their contents. - One of the key steps in DNA and RNA extraction for the purpose of subsequent analysis. ## 1- EDTA (Ethylene diamine tetra-acetate): * EDTA is a chelating agent used in lysis buffers to eliminate divalent cations (such as magnesium and calcium) from the solution. * This helps to destabilize the cell membrane, making it easier for the cells to lyse, and it inhibits DNase (degrades DNA) because it chelates mg required for the activity of DNase. ## 2. Lysozyme * Lysozyme is an enzyme that digests the peptidoglycan layer of bacterial cell walls. * In some cases, enzymes are added to the lysis buffer to help break down specific components of the cell. ## 3. Other Enzymes * **Plant cells**: cellulase to digest cellulose. * **Yeast cells**: Lyticase to degrade the cell wall of yeasts. * **Animal cells**: proteinase K to degrade proteinaceous components. ## 4. Precipitation * The desired molecule (e.g., DNA) is then precipitated out of solution using an alcohol such as isopropyl alcohol or ethanol. * This is usually done by adding the alcohol to the sample plus monovalent ions such as sodium (Na+) or potassium (K+). * As alcohol dehydrates DNA, hence it precipitates. ## 5. Washing * The precipitate is then washed to remove any contaminants. ## 6. Resuspension * The precipitate is then resuspended in a buffer solution.