HISTOLOGY

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• HISTOLOGY
• AND ITS METHODS OF STUDY

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• HISTOLOGY

• is the study of tissues
• through the examination of the architecture and
  relationship of the different types of tissue,
• through the detailed investigation of the
  structure of individual cells.

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THE FUNDAMENTAL TYPE OF TISSUE

• EPITHELIAL TISSUE
• CONNECTIVE TISSUE

• MUSCULE TISSUE
• NERVE TISSUE

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THE TISSUE ORGANIZATION

• EACH TISSUES ARE MADE OF
• CELLS
• EXTRACELLULAR MATRIX

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MAIN FUNCTIONOF THE EXTRACELLULAR MATRIX

• SUPPLIES MECHANICAL SUPPORTING FOR THE CELLS
• TRANSPORTS THE NUTRIENTS TO THE CELLS
• CARRIES METABOLITES AND SECRETORY PRODUCTS AWAY

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• The subject of histology not only deals with the
  structure of the body, but also concerns with the
  body's function.
• Histology is known as a branch of anatomy.
• Histology, in broader aspects is named the
  microscopic anatomy.

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LIGHT MICROSCOPE

• IS ONE OF PRINCIPAL INSTRUMENT
• USE IN LABORATORY

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TYPE OF MICROSCOPES

• BRIGHT-FIELD MICROSCOPE/DARK-FIELD MICROSCOPE
• CONTRAST-PHAZE MICROSCOPE
• INTERFERENCE MICROSCOPE
• POLARIZING MICROSCOPE
• FLUORESCENCE MICROSCOPE
• TRANSMISSION ELECTRON MICROSCOPE (TEM)
• SCANNING ELECTRON MICROSCOPE (SEM)
• CONFOCAL MICROSCOPE
• INVERTED MICROSCOPE

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LIGHT MICROSCOPE

• MECHANICAL PARTS
• MICROSCOPE BODY (arm, base)
• NOSEPIECE
• SPECIMEN STAGE with stage possition adjustment
• FOCUS ADJUSTMENT KNOB

• OPTICAL PARTS
• CONDENSER
• OBJECTIVES
• EYEPIECE

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OPTICAL PART OF MICROSCOPE

• The condenser
• collects and focuses light, producing a cone of
  light that illuminates the objects which are
  observed.
• Condenser has an aperture diaphragm with which
  the diameter of the light beam can be controlled.
• The more light at the specimen the better is
  resolution of the image.

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OPTICAL PART OF MICROSCOPE

• The objective lenses
• enlarged and project the illuminated image of the
  object in the direction of the eyepieces.
• The eyepiece lens
• magnifies this image and projects it onto retina
  of viewer.

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• THE TOTAL MAGNIFICATION
• IS OBTAINING BY MULTIPLYING
• THE MAGNIFAING OF THE OBJECTIVE
• AND OCULAR LENSES.

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RESOLVING POWER

• IS THE SMALLEST DISTANCE BETWEEN TWO PARTICLES AT
  WHICH THEY CAN SEEN AS SEPARATE OBJECTS.
• THE MAXIMAL RESOLVING POWER OF THE LIGHT
  MICROSCOPE IS 0.2 µm.
• THE RESOLVING POWER INFLUENCE ON QUALITY OF THE
  IMAGE.
• THE RESOLVING POWER DEPENDS MAINLY ON THE QUALITY
  OF OBJECTIVE LENS.
• THE EYEPIECES LENS THAT ONLY ENLARGE THE IMAGE
  OBTAINED BY OBJECTIVE LENS, DOES NOT IMPROVE THE
  RESOLUTION.

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LIGHT MICROSCOPE

• OBJECTIVES 5, 10, 20, 40, 60, 100 X
• MAGNIFICATION 1000 - 1500X
• RESOLVING POWER 0.2 µm

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STEPS IN PREPARATING SECTIONS FOR LIGHT MICROSCOPE

• FIXATION
• DEHYDRATION and CLEARING
• EMBEDDING
• SECTIONING
• STAINING

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FIXATION

• IS THE TREATMENT OF THE TISSUE WITH CHEMICAL OR
  PHISICAL AGENTS
• AVOID TISSUE AUTOLYSIS DIGESTION BY ENZYMES
  PRESENT WITHIN THE CELLS
• ALLOW TO PRESERVE THE STRUCTURE AND MOLECULAR
  COMPOSITION OF THE TISSUE, MAINTAINING NORMAL
  ARCHITECTURE OF TISSUE
• Therefore pieces of organ removed from body
  should be as soon as possible treated by specific
  fixatives
• FIXATIVES are the substances that stabilizing
  or cross-linking proteins, thus maintaining a
  lifelike image of the tissue.

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FIXATIVES

• COMPOSITE FIXATIVES
• BOUINs FLUID
• picric acide formalin
• CARNOYA FLUID
• ethanol chloroform acetic acide

• SIMPLE FIXATIVES
• ALDEHYDE
• neutral 4 solution of formaldehyde,
• formalin,
• glutaraldehyde
• ALCOHOLS
• ethanol (50-100)
• KETONES
• acetone (50-100)
• ORGANIC AND NONORGANIC ALCOHOLS
• acetic, picric
• HEAVY METALS
• osmium tetroxide

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POST-FIXATION STEPS

• DEHYDRATION a process removing the water from
  the tissue
• water is extracted (dehydration) from the tissue
  during its passing a greater series alcohol
  baths, beginning with 50 alcohol solution,
  progressing in stronger and stronger solution to
  100 alcohol

DEHYDRATION also replaces the fixatives with
dehydrating fluid.
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POST-FIXATION STEPS

• CLEARING is the treatment with xylene to make
  tissue transparent.
• Xylene is totally miscible with both the
  dehydrating fluid and embedding medium

CLEARING is replacing the dehydrating fluid with
the clearing fliud - xylene
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EMBEDDING

• A PROPER MEDIUM USING TO EMBEDDING
• MELTED (LIQUIDATED) PARAFFIN
• PLASTIC RESIN (for particularly hard tissue)
• POLIMERIZING RESIN epon, spure, aradite

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EMBEDDING

• Paraffin-infiltrated tissue is placed into a
  small mould,
• covered with melted paraffin,
• and allowed to cooled and harden, forming a
  paraffin block containing the tissue.

EMBEDDING PERMIT SECTIONING
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SECTIONING BY MICROTOME

• Paraffin block is mounted in a microtome.
• The microtome is the machine equipped in a sharp
  steel blade, that undercontrol of crank cuts thin
  slices of paraffin block containing tissue.
• slices are placed onto well-adhered glass slaids
• For light microscopy, the thickness of each
  section is 5 -10 µm

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UNSTAINED PARAFFIN SECTION

• Many tissue elements have approximately the same
  optical densities, therefore for light microscopy
  they have to be stained with water-soluble
  stains.

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BEFORE THE STAINING

• the paraffin must be removed from the section
  using xylene

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STAINING

• PERMIT THE EXAMINATION OF THE TISSUES BY LIGHT
  MICROSCOPE

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HISTOLOGIC STAINS

• Classes of histological stains
• Dyes stain acidic and basic components of the
  cell and extracellular matrix
• Specific dyes stains the fibrous components of
  the extracellular matrix
• Metallic salts penetrate into the tissues,
  forming metal deposits within the tissue

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LIGHT MICROSCOPE

• BASIC DYES
• Hematoxylin
• Toluidine blue
• Metylene blue
• Basic fuchsin
• ACID DYES
• Eosin
• Orange G
• Acid fuchsin

BASIC DYES HAVE AFFINITY TO ACIDIC (BASOPHILIC)
COMPONENTS OF CELL AND TISSUE ACID DYES HAVE
AFINITY TO BASIC (ACIDOPHILIC) COMPONENTS OF CELL
AND TISSUE
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HEMATOXYLIN AND EOSIN (H-E)
Dyes stain acidic and basic components of the
cell and extracellular matrix

• The most commonly use stains in histology
• Hematoxylin is a base that colors the acidic
  components of the cell a bluish tint.
• The organella - nucleus (DNA, RNA), and regions
  of the cytoplasm rich in ribosomes or another
  acidic components are stain dark blue
• The components stain with hematoxylin are
  referred to as basophilic.
• Eosin is an acid that stains the basic components
  of the cell a pinkish color.
• Most of cytoplasmic components have a basic pH
  and stain pink
• The cytoplasmic elements stain with eosin are
  said to be acidophilic.

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HEMATOXYLIN AND EOSIN (H-E)
Dyes stain acidic and basic components of the
cell and extracellular matrix
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MALLORY-AZAN

• ANILINE BLUE
• ACID FUCHSIN
• ORANGE G

H-E
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HEMATOXYLIN AND EOSIN (H-E)
Dyes stain acidic and basic components of the
cell and extracellular matrix
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SUDAN III - LIPIDS
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ORCEIN AND RESORCINE FUCHSIN
Specific dyes stains the fibrous components of
the extracellular matrix
ELASTIC FIBERS
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SILVER STAINING
Metallic salts penetrate into the tissues,
forming metal deposits within the tissue
RETICULAR FIBERS
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OsO4 STAINING
Metallic salts penetrate into the tissues,
forming metal deposits within the tissue
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PERIODIC-ACID SCHIFF (PAS)

• PAS techniques are used to demonstrate
• polysaccharides,
• neutral mucosubstances
• glycogen and carbohydrate-rich molecules
• basement membrane

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IRON HEMATOXYLIN
Metallic salts penetrate into the tissues,
forming metal deposits within the tissue
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HISTOLOGIC STAINS
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IMMUNOHISTOCHEMISTRY (IHC)

• Is the advanced and sophisticated staining method
  based on the process of localizing proteins
  within the cell/tissue by antibodies, that
  binding specifically to antigens (detected
  proteins).
• Visualizing an antibody-antigen interaction can
  be accomplished in binding with biotinylated
  secondary antibody conjugated to an enzyme, such
  as peroxidase that catalyses a colour-producing
  reaction.

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TYPE OF IHC REACTION

• ONE STEP REACTION
• Labelled primary antibody

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TYPE OF IHC REACTION

• TWO STEPS REACTION
• Primary antibody
• Secondary antibody conjugated with enzyme

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IMMUNOHISTOCHEMISTRY what for???
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IMMUNOHISTOCHEMISTRY
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IMMUNOHISTOCHEMISTRY
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FLUOROIMMUNOHISTOCHEMISTRY
Fluorescent staining methods are more sensitive
than enzyme-based staining for light microscopy
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FLUOROIMMUNOHISTOCHEMISTRY

• ONE STEP REACTION
• Primary antibody conjugated with fluorochrom

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FLUOROIMMUNOHISTOCHEMISTRY

• TWO STEPS REACTION
• Primary antiody
• Secondary antibody conjugated with fluorochrom

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ARRANGEMENTTHE TISSUE IN PARAFFIN BLOCK
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LONGITUDINAL, CROSS SLANTWISE SECTION
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LONGITUDINAL CROSS SECTION
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• IN VITRO CULTURES

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CULTURE DISHES
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CULTURE MEDIUM

• CONTAINS
• Nutritional and energy substances (AA, glucose,
  lipids)
• Vitamins (need to apropriate metabolism)
• Mineral salts (need to migration, agregation and
  adhesion)
• MEM (Minimum Essential Medium)
• DMEM (Dulbeccos Modified Eagel Medium)
• Serum (FCS - Fetal calf serum)
• growth and differentiation factors FGF, EGF,
  PDGF, IGF
• Hormons insulin and hormone binding proteins
• Peptids albumins, antiproteases
• Antibiotics
• Penicilin
• streptavidin

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PRACTICAL REASONS OF IN VITRO CULTURES

• medical research on genom of cell, chromosome
  abberation, mutation
• To study the genetical foundation of metabolic
  disorders
• Hormon and growth factors production by cultured
  cells
• To study the influence of toxins, carcinogenic
  substances and drugs/medicines on cell metabolism
  and function
• To study the immunological processes (the cell
  answer after antigen stimulation)
• To observation of the viral infection
• To make the vaccines and monoclonal antibodies
• To study the processes of organs differenciation