Presentation on theme: "Methods in Cell Biology Medical Microanatomy 602 Edie C. Goldsmith, Ph.D."— Presentation transcript:
Methods in Cell Biology Medical Microanatomy 602 Edie C. Goldsmith, Ph.D. Edie.Goldsmith@uscmed.sc.edu
Introduction The material in this power point is covered in Chapter 1 of your textbook. I would encourage you to read the text, particularly sections that are covered here. While we will not specifically cover this material during lecture, you will be expected to know this information for the exam. I can address any questions you may have during the lab period or by email. Notice that in the notes section below each slide I have added information and explanations for the images/text on that slide when necessary.
Tissue Preparation Fixation –Preserve tissue structure –Chemical Formaldehyde/glutaraldehyde - cross-linking amine groups Alcohol – denature proteins Heavy metals (osmium tetraoxide) – oxidation of proteins to form cross-links; also react well with phospholipids –Freezing Cryoprotectant (OCT, sucrose solution) Rapid low-temp freezing Lipids and activity stains (enzymes) Not all fixative works for all structures –Formaldehyde - not good for lipids Don’t over fix
Dehydration & Clearing –Prepare tissue for embedding –Removal of water can cause tissue shrinkage Infiltration –Mix of clearing agent and embedding media Embed tissue –Prevents tissue damage during sectioning –Embedding agents - Paraffin, plastic resins, acrylamide Section, mount and stain
Samples are dynamic during processing Some things get retained –Nucleic acid – protein complexes, cytoskeletal proteins, ECM proteins, lipid-protein complexes Some things get lost –Small, soluble molecules (ions, carbohydrates), glycogen, proteoglycans –Can use special fixation or staining methods
Tissue Staining Basophilic Stain with basic dye [dye + Cl - ] Toluidine blue, methylene blue, hematoxylin, alcian blue Nucleic acids, some cytoplasmic components (rRNA and rER), glycosaminoglycans and acidic glycoproteins Acidophilic Stain with acidic dye [Na + dye - ] Orange G, eosin, acid fuschin Mitochondria, cytoplasm, secretory granules, ECM proteins Dye color does not determine whether a dye is acidic or basic (see Table 1.2 in your textbook)
Hematoxylin & eosin (H&E) is the most common dye combination you will see this semester. –Hematoxylin – basic- like dye which stains negatively charged molecules (blue) i.e. Nucleic acids –Eosin - acidic dye which stains positively charged molecules (pink) Cytoplasm (proteins) Material stained with eosin Nuclei stained with hematoxylin
Metachromasia When certain dyes bind some cellular structures, their color shifts This is due to the presence of a large number of polyatomic anions (SO 4 2- and PO 4 3- ) –This leads to aggregation of the dye which changes is absorption properties –The polyatomic anions are common in ground substance (particularly cartilage); heparin granules in mast cells; rER in plasma cells Toluidine blue is an example of a dye the exhibits metachromasia. As it name would imply, this dye usually stains blue. But when it is used to stain mast cells, the granules in these cells contain lots of negatively charged molecules and the dye’s color shifts to a purple-red appearance.
Other notable stains Periodic acid-Schiff (PAS) –Carbohydrates (glycogen, mucin, basement membrane) –Periodic acid + sugar aldehyde –aldehyde + bleached basic fuchsin (Schiff reagent) magenta color PAS positive Staining (mucus)
Orcein Also known as resorcin- fuchsin stains elastin found in elastic fibers The individual fibers in this image are elastic fibers stained with orcein Aorta
Trichrome stains –Employ 3 dyes –Masson Trichrome– most common –Usually used to look at collagen in the extracellular matrix Nuclei (basophilic) – blue/black Collagen – green or blue Cytoplasm, muscle, keratin, erythrocytes – red Azan –Stains collagen, basement membrane, mucin Nuclei – red Collagen - blue nephron collagen nucleus collagen nucleus erythrocytes
Picrosirius Red Collagen specific stain Observed under polarized light the collagen appears yellowish-orange in color. Observed under normal bright field microscopy, the collagen would have a bright pink appearance. In this image of skeletal muscle, the collagen has a bright, glowing orange appearance. The striated structures are muscle cells.
Nissl This uses a basic dye on neuronal tissue. Stains the rER in neurons –Referred to as Nissl substance when observed in clumps Can also be done with methylene blue N
Gold and Silver Stains Silver stain of reticular fibers (type III collagen) String-like appearance in this image Gold staining of motor end plate in muscle Renal cortex
Lipids Preserved in frozen sections Stains used in light microscopy –Sudan black, Oil red O, Sudan IV, Osmium tetraoxide Common stain used for electron microscopy –Osmium tetraoxide Black circles in this image are lipid droplets in brown fat cells
Enzyme Histochemistry Localize enzymes within tissue Don’t over fix (frozen sections) Precipitated product Indirect method Variety of enzymes –Acid and alkaline phosphatases, dehydrogenases, peroxidases Light and EM Alkaline Phosphatase in rat kidney (Gomori stain with a lead precipitate)
Immunohistochemistry (IHC) Rely on the use of antibody directed against molecule of interest –Usually protein Direct vs indirect Amenable to many forms of detection –Fluorescence –EM (gold) –Light microscopy (color precipitate) Blue material in this fluorescent image is a cell surface protein that binds collagen.
Orange indicates sites of peroxidase reaction (lysozyme in small intestine) -amylase ab stained with Protein A - gold
In Situ Hybridization DNA or RNA distribution Target specific nucleotide probe –Radioisotope, biotin, digoxigenin Detected by autoradiography, color precipitate, fluorescence Parathyroid hormone staining (red-ish color) in Parathyroid gland
Fluorescence Microscopy Light source (UV or laser) Dyes which when excited emit in the visible region of the spectrum Confocal microscopy –Laser light source –Scan across sample and optically section –Useful on live samples (monitor real time events) –Non-destructive (in that it can be used on live cells) Easy sample preparation, fast, inexpensive
Not all fluorescent stains require an antibody Phalloidin staining of actin Acridine orange DNA – yellow; RNA - orange
Autoradiography Rely on the use of radioactive tag to follow molecule – 32 P, 35 S, 3 H Radioactive probe (amino acids, nucleotides) Specimen must be alive to monitor metabolic events Can be used with other stains Slide dipped in photographic emulsion & developed Silver grains where radioactive material is present in sample (AgBr Ag (precipitate)) Semi-quantitative
Electron Microscopy Electron beam as source ( ~ 0.005 nm) Operate under vacuum Electromagnets instead of glass lenses Detect by fluorescent screen or photographic emulsion Destructive Two major types –Transmission (TEM) and scanning (SEM)
TEM Cellular ultrastructure Bright portion – e - pass through Dark portion – e - absorbed or scattered Sections – 50 – 150 nm Fix – glutaraldehyde Stains (e - dense; heavy metals) Osmium tetroxide (OsO 4) – lipids Uranyl acetate, Lead citrate – non-specific (surface adsorbed) Ruthenium red – complex carbohydrates
Surface ultrastructure Fixed, dried, coated with gold before imaged e - reflected from surface –Results in 3-D like image SEM
SEM imageTEM image
Interpreting EMs Use internal rulers as guides –Plasma membrane ~ 10 nm wide –Nucleus between 3-10 m –Ribosomes between 15-30 nm Unique organelle features –Mitochondria and Golgi Look for largest objects first
Problems & Pitfalls Shrinkage – fixation and embedding –Appearance of empty spaces Empty spaces due to loss of material –Improper fixation and dehydration Wrinkling Precipitation of stains (may appear as dark spots all over the section) Do not memorize images - learn morphological criteria Do not rely on color
Millette’s Laws Law #1 – In any given image, if two objects look the same they are the same. Law #2 – In any given image, if two objects look different they are different.
H&E Goblet cells in Intestine PAS
A Tale of Two H&E Stains
2-dimensional appearance vs. 3-dimensional structure