How to get tissues for study. Steps in tissue preparation

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Methods to study Histology
Krishna T
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Histology

• Cell
• Tissue
• Organ
• Organ system
• Homeostasis

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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

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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

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Special situations

• Staining routine stain HE
• 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

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Basis of staining
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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

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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

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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

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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?
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Enzymatic digestion

• For the confirmation of specific substances
• Pretreatment of sections with specific enzymes
• Diastase/amylase ? for glycogen
• DNA ase ? for DNA

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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
• AB (substrate) T (trap) AT (
  reaction product) B (Hydrolyzed component of
  substrate)

enzyme
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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 Rats actin/ anti rat actin
  antibodies) ? bind with Fluorescent dye

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Monoclonal Antibodies

• Specific antigen
• (actin of rat)

Multiple Myeloma pts.
?
B lymphocytes of Immunized rabbit
Monoclonal B ells
Hybridoma cells
?
Single specific type of antibodies (Monoclonal)
( against Actin)
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Clinical Significance of Monoclonal Antibodies

• Diagnosis of tumors(tumor markers) Infections(
  HIV, Infectious Mononucleosis)
• Classify sub types (B -cell and T- cell
  lymphomas)
• Treatment Anti-TNF-a antibodies in inflammatory
  disorders

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Immunological Methods

• Immuno -fluorescence
• Direct (one step, less sensitive)
• Indirect ( more sensitive, Expensive, labor
  intensive, cant easily run in automated)
  methods
• Immunoperoxidase method
• Enzyme is used ( horse raddish peroxidase) to
  color colorless substrate into colored insoluble
  product

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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

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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

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orientation of cut
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Three dimensional picture
How you get it?
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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?
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Types Advantages of Microscopes

• 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?
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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 (cant 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 ?
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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 ?
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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
  -160C with glycerol (to prevent ice crystal
  formation)

• ? Where you find ?
• why diamond knives are used in EM?

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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?