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 Bouin’s fluid

n Picric acid

n Carnoy’s 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