Download Epithelia and Connective Tissue

Epithelia and Connective Tissue
Objective: Learn to identify the different classes of epithelial and connective tissues.
Reading: RR&K: Chapters 4-6.
Atlas pages 84-92; 116-124.
During this laboratory exercise be aware that slides of any organ will usually
display more than one epithelium and more than one connective tissue type. Skin
and GI tract are particularly useful to examine because of the variety of tissues and
epithelium present. This laboratory exercise is about identifying 9 different epithelia
and 5 different connective tissues. Try to examine at least one slide for each epithelia
and connective tissue present. You probably will not have time to examine each
slide. You can look at some of the other slides in subsequent lab exercises.
Light Microscopic Features of Various Epithelia
Epithelium
type
# of
layers
Shape of cells
Apical
specialization
Special features
Simple
squamous
1
Cells are very
flattened; nuclei
flattened or
rounded; "fried
egg shape"
None that
can be seen
in LM
Mesothelial &
endothelial cells have
flattened nuclei; thin
loops of Henle have
ovoid nuclei
Stratified
squamous, nonkeratinized
many
Surface layer
flattened; inner
layer polygonal
Usually none
visible in LM
Stratified
squamous,
keratinized
many
Surface layer
flattened, basal
layer columnar
with polygonal
cells in between
Keratinized
layer of cells
at the surface
Simple cuboidal
1
Cubic
Occasionally
microvilli &
cilia
Very common in
glandular epithelium
Stratified
cuboidal
usually
2
Cubic in both
surface and basal
layers
none
Rare; identifying
feature of sweat gland
ducts
Simple
columnar
1
Tall cells with
polygonal cross
section
Cilia &
microvilli
Stratified
columnar
usually
2
Surface cells are
tall; basal layer
cuboidal or
polygonal
usually none
visible in LM
Rare; found in
salivary gland ducts,
rectal-anal &
gastroesophageal
junctions
Pseudostratified
columnar
1
Columnar shaped
cells dominate
with short basal
layer
Cilia &
stereocilia
All cells touch basal
lamina; only some
cells reach surface
Transitional
many
Dome shaped
when organs are
empty; flattened
when they are
distended
none
Found only in urinary
tract, i.e. pelvis of the
kidney, ureter,
urinary bladder
Important: Keep in mind that the identification of an epithelium includes the
identification of any apical specialization, e.g. stratified squamous, keratinized;
pseudostratified columnar, ciliated.
I. EPITHELIUM TYPES AND LOCATIONS

Simple squamous - found predominately in four places:
o lining of the vascular system (endothelium),
o lining of visceral and pleural body cavities (mesothelium),
o Bowman’s capsule of the kidney
o alveolar spaces of the lung.
Suggested slides:
90 Jejunum; 92 Ileum; 47, 48 Aorta; 50 Blood vessels
(monkey); 52 Vena cava.

SIMPLE CUBOIDAL - found predominately in the
 lining of kidney tubules
 acini of glands,
 small ducts of exocrine glands,
 surface of the ovary and amnion vesicle
Suggested slides:
2 Simple cuboidal epithelium; 63 Axillary skin

SIMPLE COLUMNAR - found in the
 lining of stomach, gastric glands,
 small intestine,
 gall bladder,
 oviduct
Suggested slides:
90 Jejunum; 92 Ileum; 94 Small intestine composite;
133-135 Oviduct

STRATIFIED SQUAMOUS (KERATINIZED & NONKERATINIZED found in
 skin,
 oral cavity,
 esophagus,
 vagina,
 anus
Suggested slides:
63 Axillary skin, 62 Scalp, 5 Stratified squamous
epithelium, 76-78 Esophagus

STRATIFIED CUBOIDAL - Sweat gland ducts of skin
Suggested slides:
143 Mammary gland, inactive; 62 Scalp; 63 Axillary
skin

STRATIFIED COLUMNAR - largest ducts of exocrine glands, recto-anal
junction
Suggested slides:
101 Parotid gland; 102 Submaxillary gland; 103
Sublingual gland, 104 Salivary gland composite

PSEUDOSTRATIFIED COLUMNAR - found in the lining of
 trachea, bronchi,
 vas deferens, epididymis
Suggested slides:
120 Trachea; 149 Testis & epididymis; 150 Epididymis

TRANSITIONAL - Kidney pelvis, ureter & bladder.
Suggested slides:
8 Transitional epithelium; 125 Ureter, 127 Urinary
bladder
SPECIFIC SLIDE EXAMPLES
A. Small intestine
Simple columnar, simple squamous (mesothelium, endothelium), lamina propria,
dense irregular connective tissue
90D Jejunum
92A Ileum
Mesothelium surrounds the organ and can be seen very clearly in this slide. Ignore
the rounded nuclei in the next outermost band but focus on the flattened nuclei on
the very outside. These are from the mesothelium. Several large blood vessels can be
found whose lumens are lined with an endothelium.
Focus next on the luminal epithelium. This simple columnar epithelium consists of
two types of cells: absorptive enterocytes and the goblet cells. The enterocytes have a
brush border which will appear as a slightly less darkly stained band at the apical
side of the cell separated by a darkly stained line. What is this line? Underlying the
epithelium is a highly cellular connective tissue layer called the lamina propria. It
stains darkly because of the large number of nuclei. The region that is darkly
stained around the lumen is called the mucosa. It contains the epithelium, lamina
propria and a thin layer of smooth muscle (muscularis mucosae). Underlying the
mucosa is a dense, irregular connective tissue layer. At the base of the mucosa are
glands that are also made up of a columnar epithelium. What is different about the
glandular epithelium?
Ileum has the same epithelia and connective tissue as jejunum. However, the
columnar epithelium has more goblet cells but still the brush border can be seen.
Notice that in some places the epithelium looks stratified with many layers. What
causes this? Again underlying the epithelium is the lamina propria. Under the
mucosa is a relatively large layer of dense, irregular connective tissue.
B. Blood Vessels
Simple squamous epithelium (endothelium), connective tissue, elastic tissue.
48 Aorta, elastic tissue, human
This is an example of an elastic artery (Fig. 12.3, page 307). The dark lines running the
length of the section are the elastic lamina (plate 46, page 323). Examine the luminal
surface. The nuclei of the endothelial cells are stained slightly darker than the
surrounding tissue. This is probably stained with eosin and Verhoeff’s. Verhoeff's stain is
specific for the elastic laminae, which form concentric layers. There is no basophilic stain
like hematoxylin present to bring out the cellular components so the nuclei are only
slightly stained.
52A Vena cava, Masson, human
(Fig. 12.12, page 316) With Masson staining the cytoplasm appears brown whereas
connective tissue stains green. The brown stained material is mostly smooth muscle
cells. Nuclei in general are only faintly stained, but because the endothelial cell nuclei
are rather dense and flat, they are visible when viewed down their diameter. Examine the
luminal surface for elongated darkly staining structures, which are the nuclei of
endothelial cells. Also look around for smaller blood vessels. The small arteries are
collapsed which has the effect of rounding up the endothelial cell nuclei (plate 47). They
are still simple squamous.
52B Vena cava, V&E, human
This slide is stained with Verhoeff's stain to visualize the elastic fibers, and eosin to
shown the cellular structures. You will see many, dark-staining nuclei on the luminal
surface which are the endothelium. Look around for smaller vessels, called vasa vasorum
and capillaries to see if you can identify the endothelium in them. If you do not know
what a blood vessel or capillary looks like, ask your instructor. In arteries, often the
endothelium is folded or wrinkled due to contraction of the smooth muscle surrounding
the lumen.
C. Skin
Epithelia: simple squamous, simple cuboidal, keratinized stratified squamous,
stratified cuboidal
Connective tissues: dense and loose irregular, adipose
63A Ax. skin, Masson, human
Examine the large glands in the center of the section. These are apocrine sweat glands
(plate 60, page 397). The glands themselves are coiled, tubular glands with a simple
cuboidal epithelium. The ducts however, are stratified cuboidal. Both the glands and the
ducts are highly coiled which has the effect of presenting the epithelium in many
different orientations, some of which may appear to have many layers. Study the
difference in the appearance between simple cuboidal sweat glands and stratified
cuboidal (two layers of cells). Examine the epithelium at the surface (plate 59, page 395).
It is stratified squamous, keratinized. Your slide may also have sebaceous glands (plate
61, page 399) which are stratified cuboidal glands.
Immediately underlying the stratified squamous epithelium of the epidermis is a thin
layer of loose irregular connective tissue (i.e. relatively cellular with relatively small
collagen fibers). Further from the epidermis, the connective tissue becomes denser (less
cellular, larger collagen fibers). You may also find adipose tissue deep to this connective
tissue.
62C Scalp, Masson, human
62A Scalp, H&E, monkey
Both of these slides are of basically the same structures, but with different stains. In the
H&E stain, the collagen fibers are not heavily contrasted but with Masson stain they
come out a brilliant orange. See if you can find examples of stratified cuboidal sweat
gland ducts and simple cuboidal sweat glands.
64 Skin, foot, human
This is an example of thick skin where the stratified squamous epithelium is thick (but
this by itself does not define thick skin). Notice how the cells flatten out toward the
surface and how the nuclei become darker. These are dying cells. The white band
(stratum lucidum) marks the beginning of the keratinized layer.
D. Kidney
2 Simple cuboidal epithelium
This is a section from the kidney medulla and it has many tubules and blood vessels It is
stained with Mallory trichrome which makes the collagen fibers and the basal lamina
appear blue. At the edges of the section are tubules cut in longitudinal section and at the
center are tubules cut in cross section. Concentrate on the tubules with the taller
epithelium and ignore for the time being the tubules with the squamous epithelium. After
looking at the edges and the center examine the transition zone. See how the transition,
where the tubules are cut obliquely can be confusing.
124B Kidney, rabbit, PAS&H
124C Kidney; rabbit, Masson
RR&K Fig. 1.2 (page 6), Plates 93-96 (page 585-591)
These are 2 sections from the same organ but stained differently to bring our the
connective tissue. PAS&H stands for Per-iodic Acid-Schiff & Hematoxylin, the
hematoxylin being added as a counter stain to bring out some cellular details. All these
sections are oriented on the slides in the same way. With the label at the left and readable,
the cortex of the kidney runs along the upper right hand side, the medulla along the lower
left. When you mount the slide on the microscope, it will be just the reverse, i.e. cortex at
the lower left, medulla at the upper right. In this lab, we are interested in both cortex and
medulla. The kidney consists of many tubules, most of which are simple cuboidal, some
of which are simple squamous and blood vessels that are lined with a simple squamous
epithelium. PAS stains the basal lamina an intense red color. However, because the basal
lamina is very thin, it will appear as a very thin line at the base of the tubules. When
favorably oriented, you should be able to see this line. The glomeruli, which is the
filtration structure of the kidney, has a thin capsule that is lined on the inside with a
simple squamous epithelium (you can see this in Plate 1 of RR&K). This is called the
parietal layer of Bowman’s capsule. In the medulla, you will see large amounts of tubules
with a simple cuboidal epithelium and many more with a simple squamous epithelium.
There are two kinds of tubules with simple squamous epithelium, blood vessels (vasa
recta) and thin limbs of Henle. In the PAS stained slide you cannot tell the difference.
Look next at the Masson stained slide. The RBCs stain an intense red color and
cartilagenous connective tissue is an intense blue-green. The RBCs do not have their
classic biconcave shape because of preparation artifacts. The presence of RBCs identifies
the vasa recta. In Masson stain, you will notice that the basal lamina is a thin green line
under the epithelium. After you look at the tubules, try to find the minor calyx. You can
identify it by the presence of adipose tissue, and a lot of blue-green staining tissue. If
your slide is mounted correctly in the microscope, you will find the calyx at the top of the
section. Think of the calyx as a funnel. Lining the inside of the calyx is a transitional
epithelium, which you can identify by its multilayer appearance. The kidney is
surrounded by a capsule of dense irregular connective tissue which is partially preserved
along the outside of the cortex. It can be identified as the blue green material along the
lower left hand edge of the section (as you see it in the microscope).
E. Oviduct
Ciliated simple columnar epithelium, lamina propria, endothelium, irregular
connective tissue.
134 Fimbrial end of the oviduct, Masson
This organ is highly folded and the columnar epithelium consists of both ciliated and
non-ciliated cells. The cilia are just resolvable with the 40X objective. The epithelium is
classified simple columnar even though it may not appear so because of section
orientation. Identification is difficult because of the highly folded nature of the
epithelium and the highly cellular lamina propria that underlies it. Search for areas where
the epithelium is thinner. There are numerous arteries coiled through the outer parts of
the organ. If you examine these you will see well preserved examples of endothelial cells.
F. Trachea
Ciliated pseudostratified columnar epithelium, goblet cells, simple cuboidal glands.
4 Ciliated epithelium
120A Trachea
Trachea (plate 90, page 553) has several tissues. The large plates of cartilage we will
examine next week. For today, concentrate on the epithelium and the sero-mucous
glands, which may be present in some sections. These are simple cuboidal glands.
Goblet cells are everywhere in the epithelium interspersed among the ciliated cells. In
pseudostratified epithelia, there is an extra layer of cells at the base. These do not reach
the lumen but the upper layer of cells reaches the basal lamina. Hence the
pseudostratification. The cilia can be resolved at 40X by stopping down the condenser a
little.
G. Epididymis
Stereocilia, pseudostratified columnar epithelium, basal lamina, terminal bar
149 Testis & epididymis
(plate 108, page 671). Look only at the epididymis (the region pictured above) and ignore
for the time being the testis, which has a more complicated epithelium and structure. This
is a sliver stained section so the basal lamina stands out as a dark circle around the base
of the epithelium. This slide is nice for several reasons. The epithelium is well preserved
but the basal cells are sparse in some places. In some areas the terminal bar can be seen
as a dark dot at the apex of the epithelium. Individual stereocilia cannot be resolved but
they tend to fix into clumps that are visible. The basal layer of epithelial cells is sparse
and compressed down at the basal lamina. In places where the tubules are cut obliquely,
the terminal bar can be seen en face and has a darkly staining polygonal appearance.
H. Esophagus
Stratified squamous, non-keratinized; in the stomach: simple columnar
5 Stratified squamous epithelium, rabbit
79C Esophagus & stomach junction
All of these slides are of esophagus and show the non-keratinized stratified squamous
epithelium. Note the junction in 79c which shows the transition from stratified squamous
epithelium of the esophagus to simple columnar epithelium characteristic of the stomach.
The stomach epithelium is not quite as well preserved as the stratified squamous. Note
the flattened appearance of the stratified squamous epithelium near the surface and
contrast it later with transitional epithelium.
I. Mammary gland
Stratified cuboidal epithelium, dense irregular connective tissue, loose connective
tissue, adipose tissue
143C Mammary gland, inactive
The mammary glands of inactive breast consists of mainly stratified cuboidal ducts. The
glandular tissue forms under the influence of the pituitary hormone, prolactin. Notice the
difference in the connective tissue close to the ducts. This is loose, irregular connective
tissue (p. 711, fig. 22.35). It has a lower density of collagen fibers than that further away,
which is classified as dense irregular connective tissue.
J. Salivary gland ducts
Stratified columnar & simple cuboidal epithelium, adipose & loose connective tissue
102A Submandibular (submaxillary) gland, human
101B Parotid gland, human
Look at least one of these two slides. Both of these slides have a large exocrine duct with
a stratified columnar epithelium. They also have numerous smaller ducts with simple
cuboidal epithelium. Surrounding the duct is a loose irregular connective tissue.
K. Ureter
Transitional epithelium, lamina propria
8 Transitional epithelium, rabbit bladder
This is a 1.5µm thick plastic section stained with basic fuschin and methylene blue.
Fuschin is an acid dye that can be applied at different pH for different effects. The term
basic fuscin means that it was applied at a basic pH. Methylene blue is also a basic dye
and stains nuclei among other things. RBCs stain deep blue with this combination,
collagen is red, smooth muscle is deep purple-gray. The orientation of the section is
probably random so the terms top and bottom of the section with respect to the glass slide
are not too useful. Consider the top of the section, the region with the transitional
epithelium. The epithelium is relatively pale staining. Immediately underneath the
epithelium is a red staining area, which is dense irregular connective tissue. This contrast
should make it easy to find the epithelium. The high level of folding occurs because the
bladder was empty at the time of fixation. In a full bladder, the epithelium would be
straightened out. Note that the cells are rounded at all levels. This differs from stratified
squamous which has cells with flattened nuclei at the surface layers. Because the section
is so thin, not all cells show nuclei. Pan through to the other side of the tissue to the next
natural edge immediately opposite the transitional epithelium. This edge is lined with a
simple squamous epithelium, i.e. a mesothelium. Nuclei are relatively rare in this section
but you should be able to find some as you pan along the edge. The nuclei of a
mesothelium are characteristically very flat if the structure has been maintained through
fixation and handling. Note for the time being, the masses of purple staining tissue. This
is smooth muscle.
II. CONNECTIVE TISSUE
When studying connective tissues structurize the information according to:




Type of connective tissue (loose, dense, adipose, etc.)
Which cell types are present (adipocytes, fibrocytes, mast cells, etc.)
Which fiber types are present (collagen, elastic, reticular)
What is the character of the ground substance.
A. ADIPOSE TISSUE - White fat (unilocular) can be found almost anywhere. Brown
fat is usually found in animals that hibernate and in newborn infants.
9b Adipose tissue (frozen section, fat staining)
In the processing of this tissue, the fat droplet has been retained and appears a peach
color. This is unilocular fat.
9a Adipose tissue, trichrome stain
Unilocular fat has a appearance much like clumps of soap bubbles. The tissue has a very
lacy appearance because the processing has removed the fat droplet. Where the nucleus
has been retained in the section, the cell has very much the appearance of a signet ring
due to removal of the fat droplet during tissue preparation. The trichrome stain (Masson)
of this slide enhances the connective tissue.
B. LOOSE CONNECTIVE TISSUE
1. Areolar tissue
10a Areolar tissue
10b Areolar tissue
The slides are both spreads of tissue, not sections. Collagen fibers stain pink and nuclei of
fibroblasts stain blue. In sections the collagen fibers appear irregularly shaped. In
spreads, they are highly uniform. By carefully focusing this slide at higher magnifications
you can distinguish elastic fibers stained deep purple with resorcin-fuchsin. Elastic fibers
have coiled ends. Between fibers find the nuclei of fibrocytes embedded into the ground
material.
2. Lamina propria – lies under epithelia of mucous membranes
90d Jejunum
92a Ileum, c.s.
Lamina propria contains a resident population of different cell types including fibroblasts,
adipose cells and mast cells that you may be able to identify in your slides. Wandering
cells that you might find include lymphocytes and plasma cells. See if you can find any
of these and if you can sketch what you find. Mast cells have highly granular cytoplasm
(Fig. 5.17), lymphocytes have very little cytoplasm (plate 6.4), the nucleus of plasma
cells has a "cartwheel" appearance (plate 6.4) and adipocytes have a "signet ring"
appearance.
C. DENSE CONNECTIVE TISSUE
1. Regular
11b White Fibrous Tissue (tendon)
This slide reveals the fiber and cell arrangement of dense, regular connective tissue of
muscle tendon. Note how the cells are elongated and parallel to the long axis of the fibers
and how parallel the fibers. The fact that the fibers are not completely straight is a
measure of a certain elasticity in the tissue and a lack of tension on the fibers when fixed
for histology.
2. Irregular
62c Scalp, Masson, human
62a Scalp, H&E, monkey
If you have not looked at one of the skin slides by this time, examine one of these. The
connective tissue layer immediately under the surface epithelium is classified as loose
connective tissue, called the papillary layer. Immediately underneath is a layer with larger
collagen fibers, called the reticular layer, which is dense, irregular connective tissue. Note
the difference between these layers.
D. RETICULAR Tissue
12 Reticular tissue
This slide of lymph node has been stained specifically for reticular fibers, which show up
as black strands. Cellular details are very sparse. Some slides have adipose tissue
attached.
E. ELASTIC FIBERS
13 Ligamentum nuchae
This tissue has many large elastic fibers coursing through. There is both a longitudinal
and a cross section through the fibers.