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, non-keratinized 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.

Suggested slides: 90 Jejunum; 92 Ileum; 47, 48 Aorta; 50 Blood vessels (monkey); 52 Vena cava. 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:

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.