Tissue Acquisition and Preparation

I.         Tissue Acquisition and Preparation

Under anesthesia, the abdominal aorta, heart and one kidney are carefully removed from twelve rats per strain and placed in a labeled specimen cup containing approximately 75 mls of 10% Formaldehyde in phosphate buffer. The rostral end of the aorta is marked with suture to insure that the caudal end will be sectioned. The tissues are allowed to fix overnight at room temperature before they are handled. The following day, the tissues are examined and the heart and kidney are bisected before all three tissues are placed in labeled cassettes prior to automated processing. The rostral-caudal length of the hearts are measured and noted. They are then bisected at their mid-point to insure that sections will be taken from an anatomically consistent level. The kidneys are also bisected at the midsagittal plane to improve fixative penetration. The cassettes are returned to the 10% Formaldehyde solution and are held under house vacuum for a minimum of three days until they are ready to be loaded into the automatic tissue processor.

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GENERAL HISTOLOGY should help students to make SPECIFIC OBJECTIVES

SUBJECT: GENERAL HISTOLOGY
CREDITS: Total: 4.5 Theory: 2.5 Practical: 2
INTRODUCTION AND GENERAL OBJECTIVE
First year General Histology (second semester) requires students to have reached a good level of understanding of
Cell Biology and Developmental Biology. The aim of General Histology is to introduce students to the four basic
groupings that create tissues. It is essential for students to be given this general information in order to understand
the structure and function of organs and systems when studied in the second year. At the end of the year students
should be able to describe a histological preparation, indicating which types of tissue can be observed, which cell
types are present and what their respective functions are.
This guide focuses on basic tissues and gives a description of the content that students will be expected to learn.
Each topic is divided into a series of lessons that cover broadly related areas and can be considered individual

teaching units.

SPECIFIC OBJECTIVES
Topic 1
This section should help students to:
• Demonstrate knowledge of the structural and functional characteristics that define a tissue.
• Demonstrate knowledge of the mechanisms of cell differentiation, aggregation, intercellular recognition and
communication that lead to the formation of tissues.
• Demonstrate knowledge of mechanisms by which cells adapt to changes in their environment.
• Demonstrate knowledge of the nomenclature of these adaptations that will be widely used in the study of
normal and pathological structures.
• Describe the constituent elements of tissues.
• Demonstrate knowledge of the different criteria for the classification of tissues.
Topic 2
This section should help students to:
• Demonstrate knowledge of the structural and functional characteristics of epithelial tissues that distinguish them
from basic tissues.
• Demonstrate knowledge of the different types of epithelial tissue and give examples of the parts of the body in
which these can be found.
• Demonstrate knowledge of the different functions of each type of epithelial tissue and relate them to the tissue
structure.
• Demonstrate knowledge of the specialized functions of different types of epithelial cells and give examples of
the different parts of the body in which they can be found.
• Recognize the different types of epithelium in photographs or preparations and, based on the structure
identified, predict their function.
• Demonstrate knowledge of the different criteria for the classification of glands.
• Recognize certain glands in diagrams, photographs or preparations and identify the various types of gland.
Topic 3
This section should help students to:
General histology
• Name the various structural and functional characteristics that distinguish connective tissue from other basic
tissue types.
• Demonstrate knowledge of the functions of connective tissue.
• Demonstrate knowledge of the three fundamental elements that are found in all connective tissue types.
• Demonstrate knowledge of the parts of the cell involved in synthesizing components of the extracellular matrix
and how these elements are associated to one another.
• Demonstrate knowledge of the structure and function of the different cell types that are found in connective
tissue.
• Compare the different types of connective tissue according to cell types, their disposition and the relation to the
size of fibres and the extracellular matrix.
• Relate the composition of the different types of connective tissue and their specific functions.
• Demonstrate knowledge of the parts of the human body in which each type of connective tissue is found and
relate these locations to the function of the tissue.
• Recognize the types of connective tissue and their respective cells in a micrograph or preparation and describe
their functions.
• Anticipate the potential functional consequences of certain defects in connective tissue.
Topic 4
This section should help students to:
• Relate the different functions of adipose tissue to its structural characteristics.
• Describe adipose tissue as a connective tissue by relating the types and size of cells, fibres and ground
substance.
• Demonstrate knowledge of the similarities and differences between the two types of adipose tissue.
• Recognize the types of adipose tissue in a micrograph or preparation of a tissue or organ.
Topic 5
This section should help students to:
• Demonstrate knowledge of the similarities and differences between the three types of cartilage.
• Demonstrate knowledge of the functions of the three types of cartilage and relate these to their functional
characteristics and location in the body.
• Demonstrate knowledge of the stages of histogenesis and cartilage growth.
• Relate the ultrastructure of chondrocytes to their functional role in the synthesis and maintenance of the
extracellular matrix.
• Recognize the types of cartilaginous tissue in a micrograph or preparation of a tissue or organ and identify their
components, for example chondrocytes, the perichondrium, etc.
Topic 6
This section should help students to:
• Describe bone as a connective tissue in terms of cells, fibres and ground substance.
• Compare the different types of bone cells, considering their origin, structure and primary function.
• Demonstrate knowledge of the functions and physical properties of bone tissue and relate them to the specific
components of this tissue type.
• Demonstrate knowledge of the different types of bone tissue and their possible location in the body.
• Compare the two types of process of bone tissue formation, in terms of the embryonic origin of this tissue type.
• Explain the structural alterations that are produced in bone remodelation.
• Recognize the types of bone tissue in a micrograph or preparation of a tissue or organ and identify their
components, for example the Havers canals, the periosteum, etc.
• Recognize the types of limb and identify their components.
Topic 7
This section should help students to:
• Demonstrate knowledge of the three most important types of muscle tissue, compare them in terms of structure
and function, locate them in the human body.
General histology
• Demonstrate knowledge of the relations between muscle fascicles, muscle fibres, myofibrils and myofilaments.
• Explain excitation-contraction coupling. The role of T tubules and the sarcoplasmic reticulum in striated muscle.
• Recognize the mechanisms of stimulation, contraction and relaxation of muscle fibres in terms of cell activity.
• Recognize the types of muscle tissue in a micrograph or histological preparation and be able to describe their
possible function.
Topic 8
This section should help students to:
• Name the various structural and functional characteristics of nerve tissue that distinguish it from other basic
tissue types.
• Demonstrate knowledge of the structure, function and embryonic origin of the different types of cell found in
nerve tissue and give examples of where these can be found in the human body.
• Relate the structural and functional characteristics of nerve cells.
• Predict the potential effects of structural deficits on nerve function.
• Describe in detail how neurons receive, propagate and transmit signals.
• Demonstrate knowledge of the subcellular distribution of cellular organelles in neurons and identify the regional
differences in dendrites, cell body and axom.
• Demonstrate knowledge of the structure and function of the different types of synapse.
• Demonstrate knowledge of the different support or glial cells.
• Recognize the types of nerve cells a micrograph or histological preparation and be able to identify and be able
to identify the prolongations.
• Demonstrate knowledge of cerebral vascularization and its relation to human pathology.
• Demonstrate knowledge of meningeal covers and the creation, drainage and characteristics of
cephalorachidian fluid.
PROGRAMME
Topic 1. Concept, formation and classification of tissues.
Lesson 1
1.1. Concept of tissue.
1.2. Constituent elements of tissue. Cells. Fibres. Extracellular matrices.
1.3. Tissue formation. Cell differentiation. Cell aggregation. Intercellular recognition and communication. Formation
of cell communities.
1.4. Adaptation of cells to alterations in their surroundings. Atrophy. Hypertrophy. Hyperplasia. Hypoplasia. Aplasia.
Metaplasia. Dysplasia. Neoplasia.
1.5. Cell aging and cell death.
1.6. Classification of tissues.
Topic 2. Epithelial tissues
Lesson 2
2.1. General characteristics of epithelial tissues. Diversity. Basal lamina. Renewal. Blood vessels and nerves.
Embryonic origin.
2.2. Classification of epithelia. Criteria: number of cell layers (simple, stratified, pseudostratified).
Specific types: simple squamous. Simple cuboidal. Simple columnar. Pseudostratified columnar. Stratified
(keratinized, non-keratinized). Stratified cuboidal. Stratified columnar. Transitional epithelium.
2.3. Polarity and specializations of epithelial cells. Specializations of the apical membrane. Cilia. Flagella.
Microvilli. Stereocilia.
General histology
Specializations of the lateral membrane: zonula occludens. Zona adherens. Macula adherens (desmosome). Gap
junctions.
Specializations of the basal surface: basal lamina: structure and functions. Hemidesmosomes. Transport functions.
Lesson 3
3.1. Definition and classification of glands. Endocrine glands and exocrine glands. Differences between
endocrine and exocrine glands.
3.2. Classification of exocrine glands. According to the number of cells: unicellular, multicellular.
According to organization of ducts: simple, compound, straight, convoluted.
According to secretory portion: tubular, acinose, compound tubular acinose.
According to secretion mechanisms: merocrine, apocrine, holocrine.
3.3. Different types of epithelial cells. Epithelial cells specialized for transport. a) Ion transport. b) Transport by
pinocytosis.
Epithelial cells specialized for absorption.
Epithelial cells specialized for secretion: a) protein secretors, serous secretion, b) peptide secretors (APUD system,
paracrine secretion), c) steroid secretion.
Contractile epithelial cells (myoepithelial cells).
Neuroeffector epithelial cells (Merkel cells, in taste buds).
Topic 3. Connective tissue
Lesson 4
4.1. General characteristics of connective tissues. a) Functions. b) Types. c) Components. d) Extracellular
matrix. e) Embryonic origin.
4.2. Components of connective tissue. Collagen fibres: synthesis and assembly. Types of collagens. Histological
organization and morphological characteristics. Mechanical properties. Localization.
4.3. Reticular fibres.
4.4. Elastic fibres. Synthesis and assembly. Histological appearance. Mechanical properties. Localization.
4.5. Ground substance. Proteoglycans. Glycoproteins. Diffusion of substances.
4.6. Cells. Fixed cells: a) fibroblasts, fibrocytes, reticular cells; b) adipocytes.
Migratory cells: a) mastocytes; b) macrophages; c) plasma cells.
Other types of connective tissue derived from blood (lymphocytes, neutrophils, monocytes, eosinophils and
basophils).
Lesson 5
5.1. Types of connective tissue. Connective tissues: a) lax connective tissue; b) dense connective tissue; c)
dense regular connective tissue; d) dense irregular connective tissue.
Reticular connective tissue. Elastic connective tissue. Mucous connective tissue (Wharton’s jelly). Mesenchyme.
5.2. Functional histology of connective tissue. Functions: a) support; b) defence (physical and immunological);
c) repair; d) storage; e) transport.
Edema. Nutritional factors. Collagen renewal.
Topic 4. Adipose tissue
Lesson 6
6.1. General characteristics of adipose tissues. General organization. Classification of adipose tissue: white
adipose tissue, brown adipose tissue.
6.2. White adipose tissue. Differential characteristics. Body distribution. Functional characteristics: a) factors that
affect lipid uptake; b) factors that mobilize lipids. Histogenesis.
6.3. Brown adipose tissue. Differential characteristics. Body distribution. Functional characteristics. Histogenesis.
General histology
Topic 5. Cartilaginous tissue
Lesson 7
7.1. General characteristics of cartilage. Composition. Vascularization. Cells.
7.2. The three types of cartilage. Hyaline cartilage: a) composition: fibres, ground substance; b) organization; c)
histogenesis; d) growth: interstitial, appositional; e) repair; f) function and localization.
Elastic cartilage: a) composition and organization; b) histogenesis and growth; c) function and localization.
Fibrocartilage: a) composition and organization; b) histogenesis and growth; c) function and localization.
Topic 6. Bone tissue
Lesson 8
8.1. General characteristics of bone. Composition. Functions.
Bone types: a) cancellous; b) compact; c) primary (non lamellar or woven); d) secondary (lamellar); e) bone as an
organ.
8.2. Cells. Osteoprogenitor cells. Osteoblasts. Osteocytes. Osteoclasts. Bone lining cells.
8.3. Bone matrix. Organic components: a) collagen fibres; b) ground substance.
Inorganic components: hydroxyapatite crystals.
8.4. Organization. Cancellous bone. Compact bone.
8.5. Histogenesis and remodelation. Primary bone. Role of the perichondrium.
Formation of bone: A) intramembranous; B) endochondral: a) proliferation of chondrocytes; b) hypertrophy; c)
calcification. Remodelation: a) primitive medullary cavity; b) formation of secondary bone.
8.6. Microscopic and functional organization of secondary bone tissue: external circumferential systems.
Periosteum.
Haversian systems (osteons). a) Haversian Canal. b) Volkmann’s Canals.
Intermediate systems.
Internal circumferential systems. Endosteum.
8.7. Articulations: types and general characteristics.
Synarthrosis: a) synostosis; b) synchondrosis; c) syndesmosis.
Diarthrosis.
Topic 7. Muscle tissue
Lesson 9
9.1. General considerations on muscle tissue. a) Terminology. b) Specialization for contraction. c) Embryonic
origin. d) Form of muscle fibres. e) Organization. f) Types of muscle (smooth and striated).
9.2. Skeletal muscle. Histogenesis.
Skeletal muscle fibres. Myofilaments: a) thin filaments (G-actin and F-actin, tropomyosin, troponin); b) thick
filaments (myosin).
Myofibrils. a) Longitudinal and transversal sections. 2) The sarcomer. Z, M, I, A and H lines. Morphological changes
relative to the state of contraction.
Sarcoplasmic reticulum.
The sarcolemma and its relation to the extracellular matrix.
9.3. Histochemical types of muscle fibre. Red fibres. White fibres. Intermediate fibres.
9.4. Satellite cells. Skeletal muscle repair.
Lesson 10
10.1. Cardiac muscle. Histogenesis.
Cardiac muscle fibre. a) Sarcoplasmic reticulum. b) T-tubules. c) Intercalary discs: desmosomes, fascia adherens,
gap junction. d) Types of cardiac fibres: ventricular fibres, atrial fibres, conduction fibres.
General histology
Cardiac muscle repair.
10.2. Smooth muscle. Histogenesis.
Smooth muscle fibre. a) Myofilaments: thin filaments, thick filaments, dense bodies. b) Sarcoplasmic reticulum. c)
Contraction.
10.3. Types of smooth muscle fibre. Visceral smooth muscle. Vascular smooth muscle. Iris smooth muscle.
10.4. Control of smooth muscle contraction.
Topic 8. Nerve tissue
Lesson 11
11.1. General characteristics of nerve tissue. Cell types: a) neurons: structure and function; b) support cells.
Impulse conduction. Synapse. Development of nerve tissue.
11.2. Neurons. Cell body. Dendrites.
Axons: a) the axon hillock; b) initial segment; c) axoplasma and axolemma; d) terminal ramification; e) nerve
terminals; f) el transport axonal (retrograde and anterograde).
Classification of neurons. a) By arrangement and prolongations: multipolar, bipolar, pseudounipolar, unipolar. b) By
shape: pyramidal, stellate, granular, Purkinje. c) By axon length: Golgi type I and Golgi type II. d) By function:
motoneurons, sensory neurons, interneurons. e) By the nature of the liberated neurotransmitter: glutamatergic,
GABAergic, cholinergic, dopaminergic, adrenergic, peptidergic, purinergic.
11.3. The synapse. Electrical synapses.
Chemical synapses. a) The presynaptic membrane. b) Synaptic cleft. c) The postsynaptic membrane. d) Synaptic
transmission.
Excitatory synapses.
Inhibitory synapses.
11.4. The neuromuscular junction. The nerve terminal. The synaptic cleft. The postsynaptic element.
Lesson 12
12.1. Support cells of the central nervous system. Macroglia. a) Astrocytes: protoplasmic. Fibrous. b)
Oligodendroglia: myelin. Ranvier’s nodes. Saltatory conduction.
Microglia.
Ependymal cells.
12.2. Support cells of the peripheral nervous system. Schwann cells. Satellite cells.
12.3. Vascularization of the human brain. Arterial system. Capillaries. The hematoencephalic barrier. Vascular
system. Stroke mechanisms.
12.4. Meninges, ventricles and cephalorachidian liquid. Dura mater, arachnoid mater and pia mater. Meningeal
spaces. Choroid plexus. Cephalorachidian liquid. Characterization. Production. Drainage. Cellular composition.
Intracranial pressure. Clinical application of the study of cephalorachidian liquid.
LEARNING RESOURCES AND TEACHING METHODOLOGIES
Each topic consists of the theory class and the practical session, which will consist mainly of the recognition of
tissues and cells with an optical microscope and subsequent description.
The theory classes are no substitute for personal observation, interpretation and description of microscope
preparations in order to gain a better understanding of pathological reports. The faculty therefore provides a
practical laboratory equipped with binocular microscopes and monitors connected to the teacher’s microscope.
LEARNING REQUIREMENTS
Students must have sufficient preparation in Cell Biology and Developmental Biology to take the course in
Histology.

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Histology is “the study of tissue”

Histology is “the study of tissue”. This covers a rather enormous range of techniques. At its most sophisticated, histological techniques can be used to discover which genes are being transcribed in a tissue (e.g., through in situ hybridization) or what proteins are expressed (e.g., via immunohistochemistry). Histological techniques are also used to describe the microstructure of neurons (electron microscopy), the macrostructure of neurons (using, for example, the silver impregnation stain), or the connectivity of the nervous system (track tracing, Nissl staining). Other specialized techniques allow study of the time course of development of structures (e.g., 5-bromo-2'-deoxyuridine [BrdU] staining, a marker of mitosis) or evidence of their degeneration (e.g., glial fibrillary acidic protein [GFAP] staining).

While these would all fall under “study of the tissue”, histological techniques are also routinely used as accessory manipulations in lesion, pharmacological, or electrophysiological studies. An electrode recording neural activity or a cannula delivering intracerebral drugs can also inject a dye used later to verify the position of the electrode or cannula in the brain. A brain may be analyzed later to quantify the location and extent of an electrolytic or excitotoxic brain lesion. Thus, even in labs that do little tissue study per se, histological techniques are often used.

Steps of Preparing a Histological Stain of a Rat Brain

1- Perfusion: a living rat is deeply anesthetized and saline is pumped into its heart in order to clear all the blood out of the body.  Next, formalin (37% formaldehyde) is pumped through its circulatory system in order to fix the tissue.  This hardens the tissue and prevents it from decomposing.  The brain is then removed and stored in formalin until processing time. (This has already been done for us)

2- Slicing & Mounting: the brain is frozen or embedded in wax to hold its shape and then placed on a microtome in order to slice thin sections (~50 microns thick = 1/20 of a millimeter, the thickness of “fine” human hair) along some plane (coronal, sagittal, horizontal).  Typically, a coronal plane is used.  The microtome is roughly equivalent to a meat slicer.  As each slice is removed it is stored in saline or alcohol and then mounted onto a slide.  The slides have been pretreated with an adhesive (gelatin) so that the sections will stick.   

3- Staining: Process the slides through a staining procedure which involves chemically “tagging” some portion of the nervous system so that it stands out from the rest.  The specific process usually involves “hydrating” the slides (alcohols and waters), staining, dehydrating, clearing, and finally cover-slipping to protect slices from handling.  

4- Observation: Slides are examined under a microscope and evaluated for whatever anatomical parameter you are interested in.  Sections are referenced to a brain atlas in order to determine which structures are represented on each slide.  Things that are frequently assessed are white matter/grey matter, cell layers, damage such as gliosis (scarring) or track marks in the case of trying to localize a microinjection or electrode implant.

 

 Cerebellum. Using the histology websites, identify the underlined structures.

Start in the Iowa website and click in the first slide of ‘Brain, cerebellum’. In the left sidebar, click the supplemental images to see macroscopic views of the cerebellum. The surface of the cerebellum has many furrows which divide it into lobules, each of which has a superficial layer of gray matter (cortex) and a core of white matter.

Supplemental Images
Gross: normal cerebellum
Visible human: pineal gland, cerebellum
Visible human: cerebellum

Next, click ‘view 1’ in the side bar for a microscopic view. The cerebellar cortex has three layers. There is an outer molecular layer (pale pink) and an inner granule layer with many small, densely packed basophilic staining cell bodies. Between the two layers are interspersed large, prominent neurons – the Purkinje cells. The most superficial layer, the molecular layer, has few neurons, many unmyelinated nerve fibers, and many dendrites of Purkinje cells (not visible with this stain). Moving further from the surface, you will see another layer of white matter, composed of axons and dendrites going to/from the cerebellar nuclei.

Go to the Illinois website, and scroll until you find the cerebellum of the rat (w79b). Open this image and its links by clicking inside of the square, until you find the Purkinje cells. Notice their dendrites. Go back to the  Iowa website, open cerebellum – silver stain (#26) and find Purkinje cells and their dendrites by increasing the magnification power (To change the power of the image go to the  top right inset of the display)

Cerebrum.  Using the histology websites, identify the underlined structures.

Start in the Iowa website, finding the slide of ‘Brain, cerebral cortex (#23)’. In the left sidebar, click ‘Gross: normal’, for a macroscopic view of the human brain. You can see a cortex of gray matter and a central area of white matter (this is reversed from the spinal cord, where the gray matter is localized internal to the white matter). The gray matter has mostly cell bodies and dendrites, while the white matter has mostly axons and myelin. The cerebral cortex has many convolutions consisting of sulci (small grooves or valleys) and gyri (hills).

            Next, click ‘view 1’ in the side bar for a microscopic view. You’ll see two gyri separated by a vertical sulcus. To change the power of the image go to the top right inset of the display. Using a low power of 2.5x, the arrangement of neurons within the cortex gives it the appearance of having several layers. You will not be able to identify each layer but rather notice that there are layers. The third layer from the outside consists of the largest neurons (pyramidal cells) which are best observed at a power of 10x.

 In the  Iowa website, go to Cerebral cortex-silver stain (#24). In this rather beautiful slide, distinguish white and gray matter and note neuronal processes (fine black lines).

Next, go to the Illinois website, and scroll until you find the pre-postcentral cerebral cortex of a monkey (slide w78a). Once again, you will see two gyri separated by a sulcus. Open this image and its links by clicking inside of the square, until you find the pyramidal cells (aka giant betz cells). 

Still in Illinois, open slide of rat brain (w78f). Zoom by opening the links until you see the pyramidal cells with their dendrites (arrows will appear showing you where the dendrites are if you go to the top of the image and scroll to see labels).

Spinal Cord Using the histology websites, identify the underlined structures.

Go to the Illinois website, and scroll until you find the spinal cord of the rat (w80c). Look first at low magnification for overall orientation to the spinal cord, cut here in cross-section (like a salami). Note the location of the white matter relative to the gray; unlike the brain, the white matter is externally located in relation to the gray matter. The gray matter has a "butterfly" shape.[1]

In the Iowa website, find the slide of ‘spinal cord (#19): In the ventral (anterior) horn of the spinal cord, increase the magnification until you find motor neurons. These large neurons are sometimes damaged by polio. Still in Iowa, go to the slide ‘spinal cord smear: (#16) in which you can find some beautiful multipolar neurons.

Finally, go to the ‘Vertebra-disk’ slide in Iowa and click “Visible human: thoracic spine, spinal cord” in the left side bar to see a macroscopic cross-sectional view of the spinal cord (bright white structure of penny size), surrounded anterior by the vertebra and posterior by the muscle.

Slicing and mounting the tissue.  The brain will already be frozen on the microtome.  Using a slow and continuous motion, draw the blade towards yourself until the blade is fully past the brain.  Using a paint brush, gently “wipe” the slice into a Petri dish of water.  Move the blade back to the starting position away, and click the lever to raise the platform up 25 microns.  Once your group has collected enough slices, sit down and work on mounting the slices onto slides.   To do this, place the slide into the water at an angle, and using a paintbrush, carefully slide the tissue onto the slide.  Be very careful not to tear the slice, and make sure that the slice is not folded over itself.  It will probably take several attempts to do this right.

Staining the tissue and identifying the anatomical location of the slice.  Using the pre-mounted and dried slide, place the slide into the slide tray and lower into solution #1 (100% ETOH i.e., ethanol). Start the timer and move the slide into the next solution as indicated.   The only time critical and subjective steps here are the length of time in the Thionin Stain, and the length of time in the ETOH + Acetic acid.  The longer the slide is in the stain, the darker all of it will become, and the longer the slide is in the Acetic acid, the lighter the whole slide will become.  Use your judgment and comparison to the pre-stained slides to determine the approximate darkness and differential staining of grey and white matter.

Staining protocol – dried slices are taken through a hydration sequence to re-hydrate the cells.  We have already started the process for you by soaking them in 95% and then 70% ETOH.

distilled H20 – 5 min

distilled H20 – 5 min

Thionin stain – 1 min              (time is critical)

H₂0 – 10 dips

H₂0 – 10 dips

70% ETOH + acetic acid – 1-2 minutes (until white and grey matter on slices are differentiated)

H₂0 – 10 dips

H₂0 – 10 dips

At this point, the slides are taken through a sequence of progressively higher concentrations of ETOH to dehydrate them, through a couple steps of xylene to “clear” the slice in order to make tissue transparent, and then they are coverslipped.  We are going to skip these additional steps

The staining protocol will take 15 minutes, and is not labor intensive. Spend your free time reviewing what you learnt in stations C-E or, if you have not done those stations yet, becoming familiar with the websites ( Iowa ;  Illinois ).   Also, take a look at these electron microscopy images. First (click here) you can clearly see the difference between myelinated and unmyelinated fibers (make sure to click the ‘Mouse-over’ function in the top-right). This is a cross-sectional cut (like a salami). In a 3-d image, you can see the node of Ranvier, in a longitudinal view of the axon (click here). Finally, you can see the neuronal synapses (click here)

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[1] The simple reflex arc is a key concept in regard to spinal cord function. This arc begins with sense receptors in the skin. When stimulated, the impulse travels down myelinated afferent (sensory) nerve. The sensory nerve enters the spinal cord and synapses with an interneuron in the dorsal horn of the gray matter. This interneuron then synapses with a large efferent (motor) neuron in the ventral horn gray matter of the spinal cord. The impulse then travels down the myelinated axon of the motor neuron, and synapses on the motor end plate at a muscle cell. This reflex arc occurs without sending any information to the brain, and thus it can occur without conscious perception of sensation.

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Staining of histological slide preparation

Biological tissue has little inherent contrast in either the light or electron microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. Where the underlying mechanistic chemistry of staining is understood, the term histochemistry is used. Hematoxylin and eosin (H&E stain) is the most commonly used light microscopical stain in histology and histopathology. Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus; eosin, an acidic dye, stains the cytoplasm pink. Uranyl acetate and lead citrate are commonly used to impart contrast to tissue in the electron microscope.

There are many other staining techniques that have been used to selectively stain cells and cellular components. One of these techniques involves marking peripheral tumors or surgical margins, in which a certain color of dye is applied to the posterior border of a sample, another to the anterior, etc., so that one can identify the location of a tumor or other pathology within a specimen. Other compounds used to color tissue sections include safranin, Oil Red O, Congo red, Fast green FCF, silver salts, and numerous natural and artificial dyes that usually originated from the development of dyes for the textile industry.

Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. A commonly performed histochemical technique is the Perls Prussian blue reaction, used to demonstrate iron deposits in diseases like hemochromatosis.

Histology samples have often been examined by radioactive techniques. In historadiography, a slide (sometimes stained histochemically) is X-rayed. More commonly, autoradiography is used to visualize the locations to which a radioactive substance has been transported within the body, such as cells in S phase (undergoing DNA replication) which incorporate tritiated thymidine, or sites to which radiolabeled nucleic acid probes bind in in situ hybridization. For autoradiography on a microscopic level, the slide is typically dipped into liquid nuclear tract emulsion, which dries to form the exposure film. Individual silver grains in the film are visualized with dark field microscopy.

Recently, antibodies have been used to specifically visualize proteins, carbohydrates, and lipids. This process is called immunohistochemistry, or when the stain is a fluorescent molecule, immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope. Other advanced techniques, such as nonradioactive in situ hybridization, can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification (especially alkaline phosphatase and tyramide signal amplification). Fluorescence microscopy and confocal microscopy are used to detect fluorescent signals with good intracellular detail. Digital cameras are increasingly used to capture histological and histopathological image

Common laboratory stains Edit

Stain Common use Nucleus Cytoplasms Red blood cell (RBC) Collagen fibers Specifically stains
Haematoxylin General staining when paired with eosin (i.e. H&E) Orange, Cyan Blue or Green Blue/Brown/Black N/A N/A Nucleic acids—blue
ER (endoplasmic reticulum)—blue
Eosin General staining when paired with haematoxylin (i.e. H&E) N/A Pink Orange/red Pink Elastic fibers—pink
Collagen fibers—pink
Reticular fibers—pink
Toluidine blue General staining Blue Blue Blue Blue Mast cells granules—purple
Masson's trichrome stain Connective tissue Black Red/pink Red Blue/green Cartilage—blue/green
Muscle fibers—red
Mallory's trichrome stain Connective tissue Red Pale red Orange Deep blue Keratin—orange
Cartilage—blue
Bone matrix—deep blue
Muscle fibers—red

Weigert's elastic stain Elastic fibers Blue/black N/A N/A N/A Elastic fibers—blue/black
Heidenhain's AZAN trichrome stain Distinguishing cells from extracellular components Red/purple Pink Red Blue Muscle fibers—red
Cartilage—blue
Bone matrix—blue
Silver stain Reticular fibers, nerve fibers, fungi N/A N/A N/A N/A Reticular fibers—brown/black
Nerve fibers—brown/black
Fungi—black
Wright's stain Blood cells Bluish/purple Bluish/gray Red/pink N/A Neutrophil granules—purple/pink
Eosinophil granules—bright red/orange
Basophil granules—deep purple/violet
Platelet granules—red/purple
Orcein stain Elastic fibres Deep blue N/A Bright red Pink Elastic fibres—dark brown
Mast cells granules—purple
Smooth muscle—light blue
Periodic acid-Schiff stain (PAS) Basement membrane, localizing carbohydrates Blue N/A N/A Pink Glycogen and other carbohydrates—magenta
Table sourced from Michael H. Ross, Wojciech Pawlina, (2006). Histology: A Text and Atlas. Hagerstown, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3.

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