Basic Histological Techniques

BIO 408

Histology

Dr. D. L. Daley

Histology

n    Branch of anatomy that studies tissue in animals and plants

n    Histology commonly is synonymous with microscopic anatomy

n    An organism is composed of:

n    Cells, intercellular matrix and extracellular fluid or tissue fluid

n    Typically the study of histology also includes some cell biology, biochemistry, physiology and where appropriate pathology

Tissue Preparation for Light Microscopy

n    1. Fixation

n    2. Dehydration and clearing

n    3. Embedding

n    4. Sectioning

n    5. Mounting and staining the sections

Fixation

n    Fixation is the first step in any procedure in which tissue is to be preserved for histological study.

n    Fixatives kill.  They kill the tissue,as well as any bacteria that are present that otherwise would cause the tissue to rot.

n    They also coagulate or cross-link proteins, making them insoluble.

Common Fixatives

n    Buffered formalin

n   4% formaldehyde in buffered isotonic saline

n    BouinÕs fluid

n   Picric acid

 

n    CarnoyÕs fixative

 

Dehydration

n    Removal of water from the tissue and replacement with ethanol

n    A graded series of mixtures of water and ethanol are use

n   Generally from 50% - 70% to 100% ethanol

n    The fixative is also removed during the early steps of dehydration by several washes in 50% ethanol for 2 hours each

Clearing

n    100% ethanol is replaced by solvent miscible with the embedding medium

n    When using paraffin the solvent is usually xylene

n    As the tissues become infiltrated with xylene they become more transparent (clearing)

n    Typically first a mixture of 50% ethanol and 50% xylene followed by 100% xylene for an hour each

Embedding

n    Infiltration is the process by which the xylene is replaced by paraffin

n    First a 50:50 mixture of xylene (30 minutes) and paraffin followed by two changes of 100% paraffin

n    The first paraffin bath lasts for 2 hours

n    The second one is 3 hours to overnight

n   Best not to exceed 5 or 6 hours since tissue tends to shrink in the heat

n    Infiltration typically occurs in an oven at 58 -60¡C

Embedding

n    Next the tissue is oriented and embed in a paraffin block

n    Block is placed in ice water to solidify

Sectioning

n    The tissue is now embedded in paraffin and the block needs to be trimmed to a trapezoid shape and then placed in the chuck of a microtome

n    A microtome is mechanical device that advances the tissue a fixed amount (1 -10 mm) as it moves the block of tissue up and down so that the block passes over a knife that cuts the paraffin and tissue into thin sections

n    When done correctly the successive(serial) sections form a ribbon

Mounting

n    The paraffin ribbons are transferred to a storage box or directly to microscope slides that has been coated with egg albumen with the aid of a small brush

n   The albumen acts a an adhesive and sticks the sections to the slide

Mounting

n    The slides are placed on a warming tray and distilled water is added to float the paraffin sections and allow then to expand and straighten out

n    The excess water is removed and the slides dry and sections will adhere to the slides

n   Allow the slides to dry overnight

Staining

n    The sectioned tissue to be studied with the light microscope must be stained since most tissues are colorless

n    Various methods of staining have been developed to make various components of cells  and tissues

Staining

n    Most stains differentiate between the acid and basic components of the cells

n    Other stains differentiate the fibrous components of the extracellular matrix

n    Some tissues can be stained by forming metal deposits on tissue - e.g., nerve cells and certain extracellular fibers are examples

Staining

n    Basophilic stains - stain the acidic components of the cell - (e.g. DNA and RNA)

n    Hematoxylin - blue color

n    Acidophilic stains - stains many cytoplasmic elements that tend to be basic

n    Eosin - pink color

n    H&E - hematoxylin and eosin is the most used combination of stained for routine histology

Staining

n    Slides with paraffin sections on them must have the paraffin removed for staining

n    Place slides in xylene for 10 minutes

n    Next a second change of xylene for 10 minutes

n    Slides are then rehydrated through a grades series of alcohols to distilled water

n    The slides are then placed in hematoxylin for 3 to 5 minutes

n    Next place the slides in 70% ethanol for 2 to 5 minutes

Staining

n    Counter stain with eosin in 70% ethanol for 2 to 5 minutes

n    Rinse off excess eosin

n    Next dehydrate and clear in xylene

n    Add a small drop of mounting medium to the slide and finally add a cover slip

n    Allow to dry before examining

Light Microscope

n    Present day microscopes use more than one lens to enlarge the tissue and thus are called compound microscopes

n    Optical components:

n   Eyepiece or ocular

n   Objective lenses

n   Condenser

Resolution

n    Ability of a lens to show two closely spaced objects as distinct and separate rather than as one fused object

n    The maximal resolving power of a light microscope is about 0.2 mm

n   This limits useful magnification to about 1000 to 1500 times

Digital Imaging Techniques

n    Digital image capture

n   No film needed

n   Immediate visualization of acquired image

n   Digital modification including contrast enhancement

Histochemistry

n    Method of staining tissue that provides identification and localization of intracellular and extracellular macromolecules

n    Methods use enzyme activity and chemical reactivity to localize macromolecules

Histochemistry

n    Periodic acid-Schiff (PAS) reagent is common histochemical reaction performed on frozen sections

n    Stains for glycogen or carbohydrate rich molecules

n    To ensure the reaction is glycogen specific the tissue is treated with amylase

n    Thus sections treated with amylase display a magenta deposit while treated sections lack the magenta color in the same region

Immunocytochemistry

n    Use antibodies tagged with a fluorescent molecule

n   e.g. Fluorescein or rhodamine

n    First an antibody to a particular macromolecule is made and then tagged with a fluorescent molecule

Autoradiography

n    Radioactive isotopes are incorporated into macromolecules

n    Commonly tritium (3H)

n    The presence of the isotopes (and thus the macromolecule) is detected by thin layer of photographic emulsion

n    The slide is placed in the dark for several weeks and the radioactive particles emitted expose the emulsion

n    Emulsion is developed like film and then cover slipped and viewed by might microscopy

Electron  Microscopy

n    Rather than light  an electron beam is used to visualize the tissue

n    Much greater resolution because of the much smaller wavelength of an electron beam

n    Resolving power is 0.005 nm - practically only 0.2nm is possible

n    However this is 1000 times more resolution than a light microscope

n    Max magnification is about 150,000 times

Scanning Electron Microscopy

n    Used to view the surface of a solid specimen

n    Shows the 3d image of the object

n    Object must be covered with a thin layer of a heavy metal such as gold or palladium

n    Beam of electrons scans the surface and the backscatter and reflected electrons are detected and collated

SEM - Blood Clot