IN THE NAME OF GOD

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IN THE NAME OF GOD
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TEHRAN UNIVERSITY OF MEDICAL SCIENCESFACULTY OF
MEDICINEDEPARTMENT OF ANATOMYDIVISION OF
HISTOLOGY

• Lecture 1Introduction
• Dr.Hassanzadeh

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ANATOMY

• Anatomy is the science which deals with the study
  of form
• And structure of body

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SUBDIVISIONS OF ANATOMY

• 1.MACROSCOPIC ANATOMY OR GROSS ANATOMY
• 2.MICROSCOPIC ANATOMY OR HISTOLOGY
• 3.DEVELOPMENTAL ANATOMY OR EMBRYOLOGY

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HISTOLOGY

• Histology, a term derived from the Greek
• Histos,meaning tissue and
• Logia, meaning the study of
• Or knowledge
• HISTOLOGY THE SCIENCE OF TISSUES

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What is a tissue?

• A collection of cells that have similar functions
  and morphologies

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Vertebrate Tissue Types

• Epithelial
• Connective
• Muscle
• Nerve
• Many organs/structures are combinations of these
  basic types

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Organization of Tissue/Organ Includes Many Cell
Types and Patterns
Epithelium red Connective tissue green Muscle
yellow
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HISTOTECHNIQUES

• In the study of histology,
• There are two important
• Considerations with regard to methodology
• 1.The type of microscope.
• 2.The preparation of the tissue or organ in a
  manner suitable for viewing the microscope.

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METHODS

• a. Routine Methods
  b.Histochemistryc. Immunocytochemistryd.
  Autoradiographye. Stereology

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• Animal research has been directly responsible for
  most advances in medicine (human and animal) and
  will continue to be important for many years to
  come

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Units of Measure

• millimeter 10-3 meter
• micrometer 10-6 meter
• nanometer 10-9 meter

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Relative Sizes of Biological Structures

• phospholipid molecule 2 - 3 nm long
• plasma membrane 7 nm thick
• typical virus 50-100 nm diameter
• mitochondria 0.5 - 1 mm diameter
• red blood cell 7 mm diameter
• typical eukaryotic cell 12-20 mm dia
• human oocyte 150 mm diameter

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Fixation of Tissues
Light Microscopy 10 buffered formalin
Electron Microscopy
Formaldehyde/glutara
ldehyde in buffer Osmium tetroxide in buffer

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Dehydration, Embedding and Sectioning

• Light microscopy
• ethanol, xylene, paraffin
• sections 8-10 micrometers thick
• Electron microscopy
• ethanol, acetone, epoxy resin
• sections 80 nanometers thick

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Remove the water replace with wax-solvent
Imbed the oriented specimen in molten wax
50 ethanol
70 ethanol
95 ethanol
Dehydrating series
label
100 ethanol
Benzene/Xylene
paraffinwax
Miscible with ethanol dissolves wax
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After it is solid, hold the wax block cut
slices
Knife
Section
Block
Glass slide
MICROTOME - a fancy meat-slicer - holds the wax
block, cuts off thin slices, as the block is
slowly advanced mechanically
Water-bath
Mount the thin slices (sections) on slides
Lift out floating section on the slide
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For fast biopsy, imbedding is omitted - frozen
sections
Knife
Section
Block is the tissue
Glass slide
FREEZING MICROTOME holds the frozen tissue,
cuts off thin slices, as the block is slowly
advanced mechanically
Water-bath
Mount the thin slices (sections) on slides
Lift out section on the slide
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Plane of section effects
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Staining Part 1

• Dyes behave as basic or acidic molecules
• A base releases OH- an acid releases H
• Dyes form ionic bonds with tissue molecules
• Basic dyes ( charge) bind to acidic (- charge)
  tissue molecules basophilic tissue molecule
• Acid dyes (- charge) bind to basic ( charge)
  tissue molecules acidophilic tissue molecule

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Staining Part 2

• Basic dyes () include hematoxylin, toluidine
  blue and methylene blue
• Acidic dyes (-) include eosin and acid fuchsin
• Basic dyes stain RNA, condensed DNA and
  glycosaminoglycans (cartilage matrix) (-)
• Acid dyes stain cytoplasmic proteins ()

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When sections dry, remove the wax, stain the
section
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Stain with eosin - red
Wash
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Epoxy resin section (1 µm) Toluidine blue
Paraffin section (8-10 µm) Hematoxylin Eosin
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Nuclear stain showing Cells and trypanasome
parasites
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Silver stain
Pyronine stain (RNA)
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Comparison of light microscopy and transmission
electron microscopy
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Artifacts

• Artifacts are appearances not true to the
  original state of the tissue

Excise Fix (preserve) the tissue in fixative
Bruising/splitting from cutting Poor
preservation, e.g. gut lining, enzymes, lost fat
Imbed the oriented specimen in molten wax
Misleading orientation, Shrinkage distortion,
Mislabeled
After it is solid, hold the wax block cut
slices
Knife scores, chatter
Wrinkles, section not flat, splits
Mount the thin slices (sections) on slides
When dry, remove the wax, stain the section
Weak/unbalanced staining
Remove surplus stain water mount coverslip
Dirt, hair, bubbles
Dirt on lenses, bad illumination
When mounting medium has set, do microscopy
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SMEAR is another method of preparation
Drop of blood
Slide 1
Slide 2
Slide 2
On contact, slide 2 extends the drop along its 1
side
Slide 2
Pushing angled slide 2 along 1 smears the line
of blood across slide 1
Smear

• A few cells are damaged smear is not evenly
  thick staining is uneven. Same apply to SPREADS

Lift away slide 2 dry 1 stain coverslip
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GROUND PREPARATION
Lay sector flat grind thin
Cut across BONE shaft twice
Saw out a sector
Wash ground section
Dry place unstained on slide
Coverslip for viewing
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