Methods to-study-histology


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Methods to study histology

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Methods to-study-histology

  1. 1. Methods to study Histology Krishna T
  2. 2. Histology Cell Tissue Organ Organ system Homeostasis
  3. 3. How to get the histology slides? How to get tissues for study Steps in tissue preparation Fresh tissues from the body 1. fixation ◦ Formalin ( 10% formaldehyde) ◦ Osmium tetroxide for EM ◦ Mechanism - Forms cross links with proteins (Lysine) 2. Embedding – gives support for tissue slicing ◦ Paraffin or plastic resin 3. Washing & dehydration (dehydration by graded alcohols in ascending order) 4. clearing – to remove paraffin & alcohol ◦ By xylol or tulol 5. block making
  4. 4. How to get the histology slides? 6. section cutting – 5-10μ thick sections with microtome 7. mounting – on glass slide ( adhesive – albumin) 8. clearing – xylol / tulol 9. rehydrate – alcohols in descending order Staining ◦ nuclear stain – Hematoxylin ( basic stain & water soluble) ◦ counter stain – Eosin ( less water soluble but soluble in alcohol) – dehydrate in ascending order 10. Clearing – xylol / tulol 11.Mounting medium – cover glass
  5. 5. Special situations Staining – routine stain – H&E ◦ Some structures are seen/ preserved (large molecules like nucleoproteins, cytoskeleton proteins, ECM proteins- collagen, membrane proteins) ◦ some are not seen/lost (small molecules -t-RNA, large molecules like glycogen & Proteioglycans are dissolved, )during the fixation/staining process Special fixatives to retain membrane ( phospholipids) ◦ Permanganate & osmium – for EM For Elastic fibers – Orcein/ Resorcin – Fuscin For reticular fibers – Silver impregnation Histochemistry & Cytochemistry ◦ Specific binding of dye with particular molecule ◦ Fluorescent dye labeled antibody to cell component Enzyme activity Autoradiography – radio isotopes tagged with precursors of a molecule  molecule incorporated into cell/ tissue before fixation
  6. 6. Basis of staining ACIDIC DYES BASIC DYES Eosin Hematoxylin /Methylene blue Carry net negative charge Carry net positive charge React/bind with cationic With anionic components of components of the cell/tissue cell/tissue Less specific (as compared Highly pH specific with basic dyes) Acidophilic / Eosinophilic Basophilic substances (cytoplasmic filaments, ( Po4 of Nucleic acids, So4 intracellular membranous of MPS, CO proteins) components, extracellular fibers)
  7. 7. What is special about Hematoxylin? Mostly resembles basic dye but it is a mordant (helps to form links between tissue fragment & the dye) It will not dissociate in sequential staining process  unlike other basic dyes
  8. 8. Metachomasia What is it ?  Absorb certain wavelength of light and emit different wavelength Why Metachomasia ?  Polyanions of tissues bind with dye molecules result in polymer or dimers of dye molecules  appear as different color rather than expected ( methylene blue gives red or purple color) What are metachromatic substances?  Ionized So4, Po4 of cartilage Where you find it?  Mast cell granules (heparin) & rER of Plasma cells
  9. 9. PAS =Periodic Acid Schiff Special stain PAS positive substances Carbohydrate (glycogen) or carbohydrate rich molecules, Basement membrane, reticular fibers Periodic acid cleaves bond between carbon atoms  form aldehyde group Aldehyde binds with Schiff to produce magenta or pink color
  10. 10. Feulgen stain for Nuclear Proteins Acid hydrolyses or cleaves proteins from deoxyribose of DNA  leads to opening of sugar group & formation of aldehyde Schiff binds and gives magenta color to aldehyde Can be useful to quantify amount of DNA ( by using spectrophotmetry of Feulgen stained tissue) Why RNA cannot be stained by Feulgen?
  11. 11. Enzymatic digestion For the confirmation of specific substances Pretreatment of sections with specific enzymes Diastase/amylase  for glycogen DNA ase  for DNA
  12. 12. Enzyme Histochemistry Localization of enzymatic activity in tissues Best fixation – mild aldehyde ( formalin) Basis – localized reaction production of enzyme activity Used for acid & alkaline phosphatase, ATP ases enzyme AB (substrate) + T (trap) AT ( reaction product) + B (Hydrolyzed component of substrate)
  13. 13. Immuno Histo Chemistry (IHC) Antibody ( Immunoglobulin) conjugated with fluorescent dye( most common is Fluorescein) + Antigen ( foreign protein) Fluorescein  absorbs UV light and emits green fluorescence  can be seen under Fluorescent microscope (IF- Immuno Fluorescence) Example :- actin (Antigen) of Rat  infected to Rabbit  blood of Rabbit ( have poly - clonal antibodies for Rat’s actin/ anti rat actin antibodies)  bind with Fluorescent dye
  14. 14. Monoclonal Antibodies Specific antigen Multiple Myeloma pts. (actin of rat) ↓ B lymphocytes of Immunized rabbit Monoclonal B ells Hybridoma cells ↓ Single specific type of antibodies (Monoclonal) ( against Actin)
  15. 15. Clinical Significance of MonoclonalAntibodies Diagnosis of tumors(tumor markers) & Infections( HIV, Infectious Mononucleosis) Classify sub – types (B -cell and T- cell lymphomas) Treatment – Anti-TNF-α antibodies in inflammatory disorders
  16. 16. Immunological Methods Immuno -fluorescence ◦ Direct (one step, less sensitive) & ◦ Indirect ( more sensitive, Expensive, labor intensive, can’t easily run in automated) methods Immunoperoxidase method ◦ Enzyme is used ( horse raddish peroxidase) to color colorless substrate into colored insoluble product
  17. 17. Other Methods Hybridization: for localizing mRNA/DNA (NA) In Situ Hybridization: Binding ( Probe + NA) in cell/tissue FISH: If Fluorochrome is used in Hybridization technique Autoradiography: by tagging the precursor molecules (Amino acids) followed by synthesis of large molecules (NA)  localize the particular tagged molecule
  18. 18. Microscopy Resolution/ Resolving power (RP): the distance by which two objects must be separated to be seen as two objects ◦ RP of ◦ Unaided Human retina : 0.2 mm ◦ Light Microscope (LM) : 0.2 μ ◦ Electro Microscope (EM) : 1.0 nm LM: we see only two dimensional pictures, orientation of cut gives different patterns Artifacts: error in preparation process
  19. 19. orientation of cut  Click to edit Master text styles ◦ Second level ◦ Third level  Fourth level  Fifth level
  20. 20. Three dimensional picture  Click to edit Master text styles ◦ Second level ◦ Third level  Fourth level  Fifth level How you get it?
  21. 21. Types & Advantages of Microscopes 1. Phase contrast M: ◦ can see live (unstained) tissue ◦ Light passing thru denser tissue of higher refractory index  out of phase from the rest  look darker ◦ Uses : identify cells in tissue cultures ◦ Modification: Interference M: quantification of tissue masses helps in study of surface properties of cells What happens to the tissues during routine staining process?
  22. 22. Types & Advantages ofMicroscopes 2. dark Field M: special condenser illuminates specimen with strong oblique light ◦ Uses: ◦ In auto radiography ◦ Study crystals in urine ◦ Study microbes- slender spirochetes ( *Treponema pallidum ) 3. Fluorescent M: emits light in visible range when exposed to UV light ◦ Technique: filters are used between light source & specimen ◦ Naturally fluorescent substances: Vitamin “A”, Neuro- transmitters ◦ Uses ◦ Tracing pathways of nerve fibers, ◦ To detect growth markers of mineralized tissues *What is the disease caused by this bug?
  23. 23. Types & Advantages of Microscopes 4. Confocal scanning M: ◦ Conjugate with focal point of lens ◦ Computer software reconstitutes the image from the data ◦ Major difference from LM: addition of detector aperture (pin hole) ◦ Uses: can see 3D pictures 5. ultra violet M: ◦ Depends on absorption of UVL by specimen ◦ Results are recorded photographically (can’t be seen directly – why?) ◦ Uses ◦ Study of nitrogen bases ( in NA) ◦ Study amount of DNA/RNA in cells *Clinically helps in study of ploidy in tumors Highly aneuploid tumor  What is its Significance ?
  24. 24. Types & Advantages of Microscopes 6. Polarizing M: only difference is polarizer (polarizing filter) ◦ Birefringence: ability of crystalline or Para - crystalline material to rotate the phase of polarized light (double refraction) ◦ Skeletal muscle & Leydig cells ◦ *Amyloid protein: apple green ◦ ±Uric acid: negative ◦ ± ± Ca++ pyrophosphate * ,±, ± ±  clinical Significance ?
  25. 25. Types & Advantages of Microscopes 7. Electron M (EM): specimen is in vacuum ◦ Types: Transmission (TEM), scanning (SEM) ◦ Mechanism: similar to LM except that beam of electrons replace light source ◦ Recording: photoelectric plate or video detector ◦ Specimen preparation: ◦ Fixation: Glutaraldehyde (cross links with proteins), Osmium tetroxide (reacts with *phospholipids) makes cell/tissue electron dense for image enhancement ◦ Other steps are same as routine tissue processing except  Plastic is used for embedding  ± Diamond knives are used in microtome ( not metal knives) ◦ To study membranes – Freeze fracture technique {-160°C with glycerol (to prevent ice crystal formation)} • * Where you find ? • ± why diamond knives are used in EM?
  26. 26. Types & Advantages of Microscopes 7. Scanning (SEM) ◦ It differs from TEM that electron beam passes across the surface of spectrum (not thru specimen as in TEM) ◦ Resembles Television ◦ Can see 3D pictures 8. Atomic Force M: most powerful tool to study surface topography ◦ Non – optical M: works like finger tip ◦ Has highest resolution power – 50 pm ◦ *Specimen need not be in vacuum • *what is the additional advantage?