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LM specimen preparation
fixation in 10% neutral buffered formalin (formaldehyde stabilized with methanol). dehydration with alcohol, rinsing with xylene or chloroform, & infiltration with paraffin. section paraffin blocks at 6-12 um, clearing paraffin, staining. most common stain is hematoxylin plus eosin. biopsies - tissue is frozen and frozen sections cut and stained with fluorescent tagged ab.
e- beam is transmitted though a thin specimen in a manner similar to the way in which visible light is transmitted through a tissue section for LM. however, EM uses magnetic lenses to focus e-s and LM uses glass lenses to focus photons.
EM sample preparation
glutaraldehyde fixation, crosslinks proteins by forming methylene bridges bt polypeptides at reactive side groups. preserves proteins & nucleoproteins excellently, but reacts slightly with lipids
TEM sample prepartion
after glutaraldehyde fixation tissue must b post-fixed in osmium tetroxide to preserve membrane and lipid components. dehydration with alcohol and acetone and infiltration with epoxy resin. after resin curing, thick and thin sections cut on glass and/or diamond knives. sections stained with uranium and lead salts to provide contrast in microscope
microscope uses beam of e-s to scan the specimen surface (1o). as product scans across the surface of the specimen, the main by products are 2o e-s, backscatter e-s, x-rays, and photons. 2o e-s are low energy e-s emitted from the surface of the specimen and these contain the surface detail info
SEM sample preparation
fixation with gluaraldehyde and osmium tetroxide. after fixation the specimen is dehydrated in ETOH and critical point dried. after drying the sample is glued onto a specimen stub and given a conductive coating (gold, gold-palladium)
goal is to visualize some component (antigen) in a tissue section by means of an ab. polyclonal may have more sensitivity due to abs directed against several antigenic determinants. monoclonal are more specific, but there may be reduced sensitivity
section tissue, then block non-specific binding with a protein solution (bovine serum albumin, fish gelatin, etc.). incubate with 1o ab and rinse. incubate with 2o ab which contains visualization label (colloidal gold for TEM), horseradish peroxidase, alkaline phosphatase, or fluorescent label for LM
aci dyes form a salt linkage with a + charged tissue group. run of a cationic group with an acid dye = acidophilia. eosin pink stain - cytoplasm and collagen/elastic fibers
basic dye has + charge. rxn of an anionic group with a basic dye = basophilia. hematoxylin blue stain - chromatin and rER
Periodic acid Shiff stain that stains glycogen & various carbohydrate containing molecules (glycoproteins). basement membrane of Bowman's capsule in kidney in the kidney stains with PAS (magenta)
in mast cells with toluidine blue: cells with large #s of polyanions - exhibit this. mast cell granules stain red/purple with toluidine blue bc the color shift occurs due to aggregation of dye molecules
phospholipids (phosphoglycerides), glycolipids (sphingolipids), and cholesterol (sterols).
phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine are derived from glycerol. sphingomyelin dervied from serine. phosphatidylinositol is important in cell signaling. 1 & 4 enriched in outer and 2,3,&5 enriched in inner
fxn in cell-cell & cell-ECM interactions and also as receptors. diptheria toxin receptor. some cancer cells have altered glycolipids. asymetrically distributed in outer membrane leaflet probably due to attached sugar groups. present only in outer leaflet and ahve a polar carbohydrate residue that forms part of the glycocalyx
complex glycolipid with sailic acid containing oligosaccharide abundant in nervous tissue
sugar coat commonly associated with extracytoplasmic aspect of the outer leaflet of PM. may measure as much as 50nm in thickness and also known as cell coat. varies in appearance in transmission e- micrographs. may be associated with ECM components that adhere to cell surface
polar oligosaccharide side chains covalently linked to protein & some lipid (glycolipid) constituents of PM. contains cell-surface proteoglycans (heavily glycosylated glycoproteins) which consist of membrane integral proteins to which are bound glycosaminoglycans (-ly charged polysaccharides)
cellular attachment to ECM components. binding of antigens and enzymes to the cell surface. facilitating cell-cell recognition and interaction
located in both leaflets of the PM. constitutes ~2% of PM lipids. assists in maintaining membrane structural integrity by decreasing mobility of the 1st few CH2 groups on phospholipids. this may enhance permeability barrier properties of membrane. may also prevent hydrocarbon chains in membrane from coming together and crystalizing
dissolved in lipid bilayer:: contain hydrophilic & hydrophobic aas, some of which interact with the hydrocarbon tails of membrane phospholipids. may extend into both leaflets or into inner leaflet only. transmembrane proteins span entire membrane and fxn as receptors and/or transport proteins. often glycoproteins (most transmembrane). remain preferentially attached to the P-face, the external surface of the inner leaflet, and seldom remain attached to the E-face, internal surface of the outer leaflet, in freeze-fractured membranes
dont extend into lipid bilayer & are usually located on the cytoplasmic aspect of the inner leaflet. in some cells, outer leaflets possess covalently attached glycolipids to which peripheral proteins are anchored and project into intercellular space. bond to polar groups of membrane phospholipids or to integral membrane proteins. usually fxn as part of the cytoskeleton or the intracellular 2o messenger system
membrane is fluid in that some integral proteins may move within the membrane. membrane fluidity increases with rise in temp and by greater unsaturation of fatty acids in membrane. fluidity is decreased by an increase in cholesterol content.
fluorescence recovery after photobleaching can demonstrate and quantify the lateral movement of proteins and lipids within PM
transports small non polar molecules and small uncharged polar molecules (H2O via aquaporins). exhibits little specificity. occurs at rate proportional to the concentration gradient of the transported molecule across the membrane
uses channel and/or carrier proteins (CPs) specific for transported molecules. CPS highly folded transmembrane proteins undergo reversible conformational alterations results in transport of specific molecules across the membrane. CPs increase transport rate over that obtainable by simple diffusion, making the membrane permeable to molecules to which it would otherwise be almost impermeable. CPs fxn in both passive and active transport
glucose transport into RBCs
glucose will move across RBC membrane down concentration gradient via glucose transporter protein (GLUT1). stereospecific allows D but not L-glucose.
Na+-K+ PUMP fxns
pump acts to maintain constant cell volume by reducing the ion concentration & osmotic pressure intracellularly & by increasing it extracellularly-decreasing the flow of water into cell. pump plays minor role in maintenance of potential difference across PM
Na+ bind to CP induce conformational changes & unmask glucose attachment site on external side of the membrane. after glucose binds, another conformational change in the CP brings Na+ and glucose inside cell. membrane Na+K+ pump drives Na+ ions out of cell, maintaining Na+ concentration gradient. at other sites, glucose exits cell via glucose-specific Cps down chemical gradient
multidrug resistance in tumors
some tumors often resistant to a broad range of anticancer drugs bc tumor cell over-expresses an ATPase found in liver, kidney, & intestinal cells that is used by normal cells to pump toxic substances into bile, urine, or intestine. many anticancer drugs are hydrophobic molecules and tumors that over-express this 170kD ATPase pump drugs out of cell
cystic fibrosis transmembrane regulator (CFTR)
CFTR has structure closely related to MDR-ATPase. cystic fibrosis is due to defective CFTR in epithelial cell of lung and other tissues. CFTR is an ATP & cAMP sensitive chloride ion channel. in CF patients, CFTR becomes insensitive to cAMP and Cl- flux across CM is disrupted
1o glycoproteins located on cell surface that bind specific signaling molecules. control membrane permeability by regulating conformation of ion channel proteins. regulate molecule entry into cell. bind ECM molecules to cytoskeleton; these integrins are needed for cell-cell contact and cell matrix interactions. act as transducers to transfer extracellular events intracellular to 2ndary messenger systems. permit pathogenic organisms that mimic normal ligands to enter cell.
ion channel-linked receptors
binds signaling molecules that temporarily opens or closes the gate, permitting or inhibiting movement of ions across CM. includes nicotinic acetylcholine receptors on muscle-cell sacrolemma at myoneural jxn. many neurotoxins (snake venoms) affect this channel
extracytoplasmic moiety fxns as receptor & cytoplasmic aspect fxns as protein kinase. may lack extracytoplasmic moiety & as result be continuously activated
transmembrane trimeric G proteins composed of 3 polypeptide subunits. interact with G proteins after binding of signaling molecule interaction results in activation of intracellular 2ndary messengers, most common of which is cAMP and Ca
in cytosol complexed with other proteins. cortisol diffuses through CM and binds to GR in cytoplasm & releases heat shock proteins. activated GR fxns in transactivation & transexpression. GR actively transport to nucleus to activate gene expression by binding to DNA responsive elements or repress other transcription factors by binding to them & preventing them interacting with target genes.
from puffer fish that inactivates Na channels causes dizziness, tingling about mouth, respiratory failure, and death
hereditary condition due to abnormal carrier proteins that cant remove cystine from urine results in kidney stones
autoimmune disease IgG abs to TSH receptors which stimulates thyroid follicular cells. enlarged thyroid, eyeball protrusion (exophthalmic goiter)
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