HISTOLOGY INTRODUCTION

HISTOLOGY & METHODS OF STUDY

HISTOLOGY

study of tissues in the body and how these tissues

are arranged to constitute organs

histo Greek for tissue or web

Involves all aspect of tissue biology focusing on
how cell's structure and arrangement optimize
functions specific to each organ

Dependent on the use of microscopes

TISSUES

Made up of 2 components: cells & extracellular

matrix

Extracellular matrix consist of many kinds of

molecules forming complex structures
- Functions of the ECM:

1.Furnish mechanical support for cells

2.Transport nutrients to cells
3.Carry away catabolites and secretory products
4.Influence and control tissue cells
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Cells produce the ECM; usually several types of

cells make up tissues

PREPARATION OF TISSUES FOR STUDY

Tissues and organs must be sectioned to obtain

thin, translucent sections and attached to glass
slides before they can be examined

Tissue preparation must preserve the same

structure and molecular composition

- But artifacts, distortion and loss of components

are almost always present

Takes 12 hours - 2 days depending on methods

used
BASIC STEPS IN PREPARATION OF TISSUES FOR
STUDY
2.FIXATION
3.DEHYDRATION
4.CLEARING
5.INFILTRATION
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HISTOLOGY INTRODUCTION

6.EMBEDDING & SECTIONING

7.STAINING
FIXATION

Done to avoid digestion by enzymes present within

cells (autolysis) or by bacteria and to preserve the
structure and molecular composition of tissues

Can be done by chemical or physical methods

Fixatives are chemical solutions of stabilizing or
cross-linking agents that inactivate enzymes and
preserve cells structure
- Tissues need to be cut into small fragments
before fixation so chemical fixatives can fully
diffuse into cells

- Usually use 10% buffered formalin solution

DEHYDRATION

Tissues transferred through a series of increasingly

more concentrated alcohol solutions, ending in
100% which removes all water from tissues
CLEARING

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Alcohol in the tissues /cells is removed by a solution

that is miscible in both alcohol and melted paraffin
- Usually use xylene
INFILTRATION

Tissue is then placed in melted paraffin at 58*C to

allow paraffin to enter all cells/ structures of the
tissue

May also use plastic resin

Heat causes the solvent to evaporate and the
spaces within tissues fill with paraffin

EMBEDDING & SECTIONING

After infiltration, the tissue is placed in a small mold

containing paraffin and allowed to harden, forming a
paraffin block; tissue embedded in a solid medium
to facilitate sectioning

Paraffin blocks are then placed an instrument called

a microtome and are sliced by the microtome
blade into sections 1-10 micrometers thick (1
micrometer = 1/1,000 of a millimeter)

Sections are floated on water and then transferred

to glass slides to be stained
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HISTOLOGY INTRODUCTION

Tissues may also be subjected to rapid freezing

(physical fixation) and sectioned using a cryostat, a
freezing microtome
STAINING

Most tissues are colorless, thus must be stained or

dyed

Dyes stain tissues more or less selectively

- Tissue components with a net negative charge
stain more readily with basic dyes and are called
basophilic

- Cationic components have affinity for acidic dyes

and are termed acidophilic

Examples of basic dyes: toluidine blue, alcian blue,

methylene blue, hematoxylin

Examples of acidic dyes: Orange G, eosin, acid

fuschin

Most commonly used dye is a combination of

hematoxylin and eosin (H&E)

- Hematoxylin stains the DNA of the nucleus and

other acidic structures blue

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HISTOLOGY INTRODUCTION

- Eosin stains other cytoplasmic components and

collagen pink

Other staining methods:

1.Periodic acid and Schif reagent (PAS) better DNA and polysaccharide staining
2.Trichrome stains - better ECM visualization
3.Lipid soluble dyes - oil red O; Sudan black
4.Metal impregnation techniques - silver salts

Counterstain is a single stain that is applied by

another method to allow better recognition of nuclei

or other structures

Final step is mounting a protective glass coverslip

on the slide with adhesive mounting media

LIGHT MICROSCOPY
Bright-field Microscopy

Stained preparations are examined by means of

ordinary light that passes through the specimen

Microscope is composed of optical and mechanical

parts
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Optical component consists of 3 systems of lenses:

condenser, objective, eyepiece

Condenser collects and focuses light, producing a

cone of light that illuminates the object to be
observed

Objective lenses enlarge and project the

illuminated image of the object in the direction of

the eyepiece

Eyepiece/ ocular lens further magnifies the image

and projects it onto the viewer's retina

Total magnification is obtained by multiplying the

magnifying power of the objective and ocular lenses

Resolving power is the critical factor in obtaining a

crisp detailed image; defined as the smallest

distance between two particles at which they can be
seen as separate objects
- The maximal resolving power of a light
microscope is 0.2 um

- The resolving power of a microscope depends

mainly on the quality of its objective lens

Digital cameras, video cameras and image

enhancing programs
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HISTOLOGY INTRODUCTION

Fluorescence Microscopy

When certain substances are irradiated by light of a

proper wavelength, they emit light with a longer
wavelength ( fluorescence)

Tissues are irradiated with ultraviolet (UV) light and

the emission is in the visible portion of the spectrum

The fluorescent substances appear brilliant on a

dark background

The microscope has a strong UV light source and

special filters that select rays of different
wavelengths emitted by the substances

Fluorescent stains include: acridine orange (bind to

DNA and RNA), Hoechst stain (bind to DNA)

Fluorescent compounds may also be coupled with

antibodies that specifically bind to certain cell
components

Phase-Contrast Microscopy & Diferential

Interference Microscopy

Unstained biological specimens are usually

transparent since all parts have almost the same

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HISTOLOGY INTRODUCTION

optical density, thus phase-contrast microscopy is

used

Phase-contrast microscopy is based on the

principle that light changes its speed when passing
through cellular and extracellular structures with
different refractive indices
- These changes cause structures to appear
lighter or darker in relation to each other

- Does not require fixation or staining thus allows

observation of living cells and tissue cultures

Diferential interference microscopy produces an

image with a more apparent 3-dimensional aspect

ELECTRON MICROSCOPY
Transmission Electron Microscopy

TEM is an imaging system that permits resolution

around 3 mm (high resolution) allowing
magnifications of up to 400,000 times to be viewed
with detail

Applies only to isolated molecules or particles

A beam of electrons is deflected by electromagnetic
fields similar to light deflection in glass lenses

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HISTOLOGY INTRODUCTION

Scanning Electron Microscopy

SEM allows pseudo 3 dimensional views of the

surfaces of cells, tissues and organs

Like TEM, this uses a narrow beam of electrons

CELL & TISSUE CULTURE

Live cells and tissues can be maintained and

observed outside the body (in vitro)

Cells and tissues are grown in complex solutions of

known composition (salt, amino acids, vitamins) to
which serum components and specific growth
factors are added

Primary cell culture - dispersed cells cultivated in

a clear dish to which they adhere as a single layer
of cells

HISTOCHEMISTRY & CYTOCHEMISTRY

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HISTOLOGY INTRODUCTION

Indicate methods for localizing cellular structures in

tissue sections using unique enzymatic activity
presents in those structures

Usually applied on unfixed or mildly fixed tissue

(enzymes must be preserved); thus often sectioned
by cryostat

Procedure:
2.Tissue sections immersed in a solution that
contains the substrate of the enzyme to be
localized
3.Enzyme allowed to act on its substrate
4.Section is placed in contact with a marker
compound
5.This marker compound reacts with a molecule
produced by the enzymatic action on the
substrate
6.The final reaction product (which is insoluble) is
visible on light or electron microscopy when
stained

Examples of enzymes that can be detected

histochemically: phosphatases, dehydrogenases,

peroxidase

IMMUNOHISTOCHEMISTRY
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A specific macromolecule present in a tissue

section may be identified using tagged compounds

that specifically react with that macromolecule
- The compound that interacts with the molecule to
be identified must be tagged with a label that can
be detected under light or electron microscopy

- Labels may be fluorescent compounds,

radioactive atoms, peroxidase or other enzymes

or metal

A tissue section is incubated in a solution

containing an antibody to a protein of interest

- If the protein is present in the tissue, the antibody
binds specifically to the protein

- Antibodies are tagged with fluorescent

compounds, peroxidase or alkaline phosphatase

- Antibodies are either polyclonal antibodies

(capable of binding to different regions of the
protein to be identified) or monoclonal
antibodies (binds only to a specific region of
protein to be identified)
HYBRIDIZATION TECHNIQUES
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Permit analysis of the molecules involved in the

process of information flow from DNA to protein

Is the binding between two single strands of nucleic

acids (DNA with DNA, RNA with RNA, or RNA with
DNA) that recognize each other if the strands are
complementary
- The greater the similarities of the sequences, the
more readily complementary strands form
"hybrid" double stranded molecules

Allows the specific identification of sequences of

DNA or RNA

Used in determining if a cell has a specific

sequence of DNA, identify cells containing specific
mRNA or determining the localization of a gene in a
specific chromosome

PROBLEMS IN THE STUDY OF TISSUE SECTIONS

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Artifacts are minor structural abnormalities

produced during tissue preparation

Includes:
- shrinkage of cells producing artificial spaces
between cells

- loss of molecules like fat or glycogen

- wrinkles of the section
- precipitates of stains
A 3-dimensional tissue is processed into slides, but
the tissue sections are only 2- dimensional

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