Techniques Flashcards
Fixatives used for routine microscopy…
Buffered isotonic solution of 4% formaldehyde or 2% glutaraldehyde
React with amine groups (NH2) of tissue proteins.
Glut: because it is a dialdehyde, which can cross-link proteins
Osmium Tetroxide (osmic acid)
2nd part of a double fixation procedure (glutaraldehyde being the 1st step) has become the standard procedure in preparations for ultrastructural studies.
Tetrahedral structure and nonpolarity allow it to penetrate charged cell membranes.
Stabilizes proteins w/o destroying structural features, binds to phospholipid head regions and prevents coagulation during alcohol dehydration
Basophilic
Tissues that stain more readily with basic dyes. Tissue components that ionize (create electrostatic salt linkages) and react with basic dyes do so because of acids in their composition.
Examples of basic dyes
Toluidine blue
Methylene blue
Hematoxylin
Basophilic tissue components
Nucleic acids, glycosaminoglycans, acid glycoproteins
Acidophilic
Dyes that stain acidophilic components such as mitochondria, secretory granules and collagen
Example of a acidophilic dye
Eosin
Resolving power
The smallest distance between two particles at which they can be seen as separate objects the quality of the image– it’s clarity and richness of detail– depends on the microscopes resolving power.
Resolving power of a light microscope
~ 0.2 um
Resolving power of electron microscope
0.1 nm although in practice 3nm is the best
Chemical fixation
Tissue samples are immersed in solutions of stabilizing or cross-linking agents called fixatives to avoid tissue digestion by enzymes present within the cells, or by bacteria to preserve the structure and molecular composition
Primary cell cultures
Cells that are cultured directly from a subject. Most have a limited lifespan. Cells may be released from soft tissues by enzymatic digestion such as collagenase, trypsin or pronase which break down ECM
Immortalized cell line
A cell line that has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification. This process is called transformation and may convert a normal cell into a cancer cell
Periodic acid-Schiff (PAS) reaction
Stain for polysaccharides like glycogen in the liver
Phalloiden
Stains actin when conjugated with fluorescent dyes
Polyclonal Antibodies
When several groups (clones) of B lymphocytes of the animal that was injected with protein X recognize different parts of protein X and each group produces an antibody against each part.
Monoclonal antibody
Individual lymphocyte clones (B lymphocytes fused with tumor cells i.e. Hybridomas) can be isolated in culture separately so that different antibodies against protein X can be collected separately.
Advantages to using a monoclonal antibody vs a polyclonal antibody
Monoclonal can be selected to be highly specific and to bind strongly to the protein to be detected. Therefore there is less specific binding.
Hybridomas provide immortal cell lines with ability to produce unlimited quantities of specific antibodies
Advantages to using a polyclonal antibody vs a monoclonal antibody
Can help increase signal produced by target protein as antibody can bind to more than one epitope
Less sensitive to antigen changes
Useful when nature of antigen is unknown
Direct method of immunocytochemistry
The antibody must be tagged with an appropriate label.
Ex: fluorescent compound, gold particles
Indirect method of immunocytochemistry
1) antibodies to protein of interest (human) must be produced in an animal of another species (eg. rat)
2) immunoglobulin from a noninjected rat must be injected into the animal of a 3rd species (eg. Goat) in order to induce antibody production to rat immunoglobulin
3) these goat antiantibodies (fluorescently conjugated) bind to the rat antibody that has previously recognized the human protein that can then be visualized.
Gel Electrophoresis/Western Blot
Isolation of Proteins
1) mixtures of proteins are obtained from homogenized cells and treated with a strong detergent (Sodium dodecly sulfate) and mercaptoethanol to unfold and separate the protein subunits
2) samples are put in wells of a polyacrylamide gel and submitted to an electric field, with proteins migrating along the gel according to size and shape.
3) a mixture of of proteins of known sizes is added to a well as a reference to identify molecular mass of other proteins
Detection & Identification of Proteins
1) all proteins stained the same color (bromophenol blue); color intensity is proportonal to protein concentration.
2) proteins can then be transferred from the gel to a nitrocellulose membrane. The membrane is incubated with a labeled antibody made against proteins that may be present in the sample (immnoblot)
Distortions & Artifacts caused b tissue processing
Shrinkage produced by fixative or ethanol
Artificial spaces due to loss of molecules (glycogen and lipids)
Precipitates of stain
Macrophage and neutrophil metabolism shift while in an anoxic, inflammatory environment
Oxidative to glycolysis
Integrins
Proteins of the plasma membrane that are linked to cytoplasmic cytoskeletal filaments and to extracellular molecules allowing for constant exchange of influence both ways
Membranes are only visible using _________ because __________
Electron microscope
They range from 7.5 to 10 nm in thickness
___________ breaks up the close packing of the phospholipid long chains, and this disruption makes the membrane more fluid
Cholesterol
Proteins make up __% of the plasma membrane
50%
Proteins of the plasma membrane include:
Integral Proteins: directly incorporated within the lipid bilayer
Peripheral Proteins: exhibit a looser association with membrane surfaces
Receptors
Glycoproteins and glycolipids with carbohydrate moieties that project from the external surface of the plasma membrane and participate in important interactions such as cell adhesion, recognition, and response to protein hormones
Fluid-Phase Pinocytosis
Small invaginations of the cell membrane form and entrap extracellular fluid and anything in solution fluid. Most fuse with lysosomes but some move to the surface opposite their origin.
“Cell-drinking”
Receptor-Mediated Endocytosis
Binding of a ligand with high affinity to its specific receptor causes widely dispersed receptors to accumulate in coated pits.
The major polypeptide of coated pits
Clatherin
Endosomes are acidified by
ATP-driven H+ pumps
Phagocytosis
Mechanism by which cells incorporate and remove foreign bacteria, Protozoa, fungi, damaged cells, and unneeded extracellular constituents via a phagosome
Exocytosis
The term used to describe the fusion of a membrane-limited structure with the plasma membrane, resulting in the release of its contents into the extracellular space without compromising the integrity of the plasma membrane.
An increase in cytosolic Ca+ often triggers Exocytosis
G proteins bind to…
… Guanine nucleotides
Activation of G protein
First messenger binds to a receptor inducing conformational changes, activating the G-protein–guanosine diphosphate complex
A guanosine diphosphate-guanosine triphosphate exchange releases the alpha subunit of the G protein, which acts on other membrane-bound intermediaries called effectors
The effector is an enzyme that converts an inactive precursor molecule into an active scone messenger, which can diffuse through the cytoplasm and carry the signal beyond the cell membrane, inducing a cascade
Endoplasmic Reticulum
A continuous system of membranes, intercommunicating channels and sacs which encloses a space called a cisterna in eukaryotic cells
In many places, the cytosolic side is covered by polyribosomes synthesizing protein molecules, which are then injected into the cisternae (rough)
The principle function of the RER
To segregate proteins not destined for the cytosol.
Initial (core) glycosylation of glycoproteins
Synthesis of phospholipids
Assembly of multichain proteins
Post-translational modifications of newly formed polypeptides
Functions of SER
Oxidation, conjugation and methylation processes employed by the liver to degrade certain hormones and neutralize noxious substances.
Synthesis of phospholipids for all cell membranes
Function of the Golgi Complex
Completes post-translational modifications and packages and places an address on products that have been synthesized by the cell.
Condensing vacuoles bud from the Golgi cisternae, generating vesicles that will transport proteins to various sites
Glycosylation, sulfating, phosphorylation and limited proteolysis of proteins
Initiates packing, concentration, and storage of secretory products
Lysosomes
Sites of intracellular digestion and turnover of cellular components.
Membrane-limited vesicles that contain a large variety of hydrolytic enzymes whose main function is intracytoplasmic digestion
Lysosomal enzymes
Acid phosphatase Ribonuclease Deoxyribonuclease Proteases Sulfatases Lipases beta-glucuronidase
________ are synthesized and segregated in the ________ and subsequently transferred to the Golgi complex, where the ________ are modified and packaged as lysosomes.
Lysosomal enzymes
RER
enzymes
Primary Lysosomes
Lysosomes that have not entered into a digestive event.
Fuse with phagosome and empty their hydrolytic enzymes into the vacuole, becoming a secondary lysosome
Secondary Lysosome
When primary lysosomes fuse with phagosome and acidify the vacuole.
After digestion, nutrients diffuse through the lysosomal-limiting membrane and enter the cytosol.
Residual bodies
Indigestible compounds that are retained within the vacuoles
In some long-lived cells, large quantities of residual bodies accumulate as lipofuscin.
Melanosis coli
Disorder of pigmentation of the wall of the colon as a result of lipofuscin in macrophages.
Lipofuscin also seen in lymph node macrophages in wistar rats
Autophagosomes
Secondary lysosomes that fused with membrane-enclosed organelles or portions of the lysosome during turnover of cytoplasmic organelles.
Cytoplasmic digestion by autophagosomes is enhanced in secretory cells that have accumulated excess secretory product.
Digested products of lysosomal hydrolysis are recycled by the cell to be reutilized by the cytoplasm.
Proteasome
Multiple-protease complexes that digest proteins targeted for destruction by attachment to ubiqitin
Function of Proteasome
Orchestrate protein degradation to remove excess enzyme an other proteins that become unnecessary to the cell after they perform their normal functions or incorrectly folded proteins
Degrades viral protein
Degrades individual proteins vs whole organelles like lysosomes
Ubiquitin
Small protein found in all cells that targets proteins for degradation.
Steps of Ubiquitin Degradation
A Ubiquitin molecule binds to a lysine residue in the protein to be degraded
Other Ubiquitin molecules attach to the first one
The complex is recognized by the regulatory particle that contains ATPase
The protein is unfolded by the ATPases using ATP as energy
The protein is translocated into the core particle, where it is broken into peptides of about 8 amino acids each
The peptides are transported into the cytosol where they are broken down to amino acids by cytosol enzymes
Apoptosis
Programmed cell death
Signs of an apoptotic cells
Compact, dark-stained nucleus (pyknotic nucleus)
Chromatin is cut into pieces by DNA nucleases (TUNL assay)
Blebs (cytoplasmic large vesicles) detach from the cell surface
Blebs remain within the plasma membrane so that it can be readily phagocytosed by macrophages
Goblet cell
Mucus secreting cell of the gut and lung
Contains strongly hydrophilic glycoproteins called mucins
Secretory granules fill the extensive apical pole of the cell, with the nucleus at the base which is rich in RER and a well-developed Golgi complex
Glycosyltransferases
Enzymes in goblet cells found in the RER and Golgi complex that add monosaccharides to core proteins to create mucins/glycoproteins
Connective Tissue
Provides a matrix that connects and binds the cell and organs and ultimately gives support to the body
The major constituent being ECM (protein fibers and ground substance)
Three classes of connective tissue components
Cells
Fibers
Ground substance
Ground substance
Highly hydrophilic, viscous complex of anionic macromolecules (glycosaminoglycans & proteoglycans) and multiadhesive glycoproteins (laminin, fibronectin) that imparts strength and rigidity to the matrix by binding to receptor proteins (Integrins) on the surface of cells and to other matrix components
The _________________ is the medium through which nutrients and metabolic wastes are exchanged between cells and their blood supply
Connective tissue matrix
