Histology Laboratory

Prelim notes

HISTOLOGY

study of the tissues of the body and how these tissues are arranged to constitute organs.

Tissues 2 interacting components:

Cells and Extracellular matrix

Extracellular matrix

consist of many kinds of macromolecules. It support the cells

TISSUE PREPARATION

Tissue samples are prepared via series of steps eventually creating a tissue block which will be cut into thin slices, and mounted on slides for study \n

FIXATION

to preserve tissue structure the tissue sample are soaked or the solution is introduced via blood vessels

Light Microscope

37% Formaldehyde

Electron Microscope

Glutaraldehyde

stabilizing the protein in tissue

Reacts with the amine (𝑁𝐻2) groups of proteins

DEHYDRATION

removal of water \n • Soaked in different alcohol concentrations ending in 100%

CLEARING

clearing of alcohol \n • Soaked in organic solvent solution in which both paraffin and alcohol are miscible

INFILTRATION

soaked in melted paraffin; epoxy resins for tissue samples for EM

EMBEDDING

placed in tissue cassette to allow hardening of the sample

TRIMMING

paraffin block is trimmed to expose tissue for sectioning

STAINING

the process of dipping the sample into different chemical to use in distinguishing histological elements

Hematoxylin

a basic dye; typically colored purple to blue

basic dye

binds to acidic components of a tissue, which are thus said to be "basophilic."

Eosin

an acidic dye; typically colored pink to red

acidic dye

binds to basic components of a tissue, which are thus said to be “acidophilic oe eosinophilic.“

Different light microscopy

bright-field, flourescence, phase-contrast

Bright-field microscopy

Uses ordinary light; tissue color is due to staining with dyes

Fluorescence microscopy

Uses UV light; at specific wavelength, light excites the fluorescent molecules emitting light

Phase-contrast microscopy

Uses different refractive index; allow examination of cells without fixation or staining

ELECTRON MICROSCOPY

Based on the interaction of tissue components with beams of electrons

Different electron microscopy

Transmission, Scanning

Electron-lucent areas (bright)

Electrons pass readily

Electron-dense areas (dark)

Electrons were absorbed or deflected

Scanning Electron Microscopy

gives 3D view

Cell and tissue culture

Cells are grown in vitro (outside the body)

Enzyme histochemistry

Enzyme activity is observed in locations where substrates can be detected. Frozen tissues are sectioned with cryostat because fixation and paraffin denatures enzymes (protein denaturation)

Enzyme classes

\n phosphatases, dehydrogenases, peroxidases

Immunohistochemistry

Reaction with antigen and antibodies labeled with visible markers (e.g. fluorescent compounds or peroxidase in LM, gold in TEM)

Magnification

The ability of the microscope to enlarge an image.

Resolution

The ability of the lens to distinguish two points as clear and as distinct. The degree to which a microscope can distinguish fine details

Parts of microscope

eyepiece, objective lenses, arm, nosepiece, base, stage, stage clip, iris diaphragm, light source, condenser, course, and fine adjustment knobs, stage adjustment knobs

Eyepiece (Ocular Lens)

The part that is looked through at the top of the compound microscope.

Objective Lenses

3-5 optical lens objectives; 4x, 10x, 40x, and 100x are the most common magnifying powers

total magnification

Objective lens magnification x ocular lens magnification

Scanner

40X

LPO

100X

HPO

400X

Oil immersion objectives

1000X

Arm

Supports the microscope head and attaches it to the base.

Nosepiece (revolving)

Nosepiece (revolving)

Base

Bottom base of the microscope that houses the illumination & supports the \n compound microscope.

Stage or Platform

The platform upon which the specimen or slide are placed.

Stage clips

Clips on the stage that hold the slide in place on the mechanical stage.

Iris Diaphragm

• Circular opening in the stage where the illumination from the base of the \n compound microscope reaches the platform of the stage. \n • Control the amount of light received by the condenser

Condenser

This lens condenses the light from the base illumination and \n focuses it onto the stage.

Coarse and fine adjustment knobs

Adjusts the focus of the microscope.

Carry the microscope

upright with two hands

Inspect microscope

before use

Clean lenses

with proper cleaning tools

push the microscope across the table

Don’t

Unplug

carefully

Return microscope

properly

Always begin viewing every slide using

the scanning power (4x) objective

When focusing with the high-powered objective

do not use the coarse adjustment knob.

Adjust the iris diaphragm

This will improve contrast, providing a a clearer view of the specimen

Most organs can be divided into

Parenchyma, Stroma

Parenchyma

Made up of cells that are responsible for the organ’s specialized functions

Stroma

• Made up of cells that have a supporting role in the organ \n • It is always a connective tissue in all organs, except in the brain and spinal cord

High cellularity

Numerous cells that are closely packed together with minimal intervening material

Avascular

Relies on receiving oxygen and nutrition from underlying connective tissue (where blood vessels are found)

Exhibits polarity

Apical pole, lateral surface, basal pole

Apical pole

faces open space

Lateral surface

attachment to neighboring cells

Basal pole

attached to basal lamina

Epithelial cells

polyhedral cells, closely packed, with minimal intervening substance

Epithelial cell shapes

squamous, cuboidal, columnar

Epithelial cell nuclei

oval, spherical, flattened

Importance of epithelial cell nuclei

• Indicator of cell shape and density \n • Determines the number of cell layers

Basement Membrane

the entire structure beneath the epithelial cells, as seen on LM

Basal Lamina

• An ultrastructure; trilaminar as seen on EM \n • Its macromolecules are a product of the epithelial cells \n • E.g. Type IV collagen, Laminin

Basal Lamina macromolecule

Type IV collagen, Laminin

Reticular Lamina

Its macromolecules are a product of the connective tissue cells

Reticular Lamina macromolecule

Type III collagen, Type VII collagen

FUNCTIONS OF EPITHELIUM

• Covering, lining, and protecting surfaces \n • Absorption \n • Secretion

Function of epithelium specialized cells

• Contractile (myoepithelial cells) \n • Specialized sensory cells (taste buds, olfactory epithelium)

INTERCELLULAR ADHESION & JUNCTIONS \n (LATERAL AND BASAL MODIFICATIONS)

Closely packed organization is made possible by adhesion and \n communication between cells

INTERCELLULAR ADHESION & JUNCTIONS

• Tight (occluding junctions)

• Adherent (anchoring junctions)

• Gap junctions

Tight (occluding junctions)

Form a seal between adjacent cells

Adherent (anchoring junctions)

Sites of strong cell adhesion

Gap junctions

Channels for communication between adjacent cells

Tight junction (Zonula Occludens)

* Occludins, daudins
* Actin Filament
* Seals adjacent cells to one another
* compromise the fetal blood-brain barrier leading to neurologic disorder

Adherens junction (Zonula adherens)

* e-cadherin
* Actin filaments
* provides points linking the cytoskeleton of adjacent cell
* loss of e-cadherin in epithelial cell tumors promotes tumor invasion

Desmosomes (Macula Adherens)

* desmogleins
* intermediate filament (keratin)
* strengthening the tissue
* Autoimmunity against desmoglein I leads to a dyshesive skin disorder

Hemidesmosome

* integrins
* intermediate filament
* Anchors the cytoskeleton to the basal lamina
* mutation in the integrin gene can lead to epidermolysis bullosa, skin blistering disorder

Gap junctions (Nexus)

* connexin
* No cytoskeleton component
* Allows direct transfer of small molecules and ions from one cell to another
* mutation in the connexin gene can lead to deafness and peripheral neuropathy

SURFACE MODIFICATIONS (APICAL)

• Microvilli \n • Stereocilia \n • Cilia

Microvilli

Finger-like extension or processes; non-motile

Appears as a brush border or striated border under the LM

Coated by glycocalyx on their surface

Microvilli core

actin filaments

Microvilli function

increase surface area for absorption

Stereocilia

Longer than microvilli; non-motile

Stereocilia Core

actin filaments

Stereocilia Functions

• Absorption and sensory \n • Lines the epididymis and ductus deferens \n • Inner ear sensory cells \n • Hair cells (auditory and vestibular perception)

Cilia

• Long, motile; larger and longer than microvilli \n • Abundant on cuboidal or columnar cells \n • Can be distinguished under the LM \n • Present in the apical surface of cells specialized for transport \n of fluid or mucus over the surface of the epithelium

Cilia Core

microtubule (axoneme)

GENERAL CLASSIFICATION OF EPITHELIUM

• Surface Epithelium \n • Glandular Epithelium

Surface Epithelium

• Simple Epithelial Tissues \n • Simple squamous epithelium \n • Simple cuboidal epithelium \n • Simple columnar epithelium \n • Ciliated columnar epithelium \n • Stratified Epithelial Tissues \n • Stratified squamous epithelium \n • Stratified cuboidal epithelium \n • Stratified columnar epithelium \n • Pseudostratified Columnar Epithelium \n • Ciliated pseudostratified columnar epithelium \n • Transitional Epithelium

Simple Squamous Epithelium location:

the lining of lung alveoli; parietal layer of Bowman’s capsule in the kidneys