Objectives:

Students should be able to explain the meaning of the terms listed at the end of this exercise. The structural features that facilitate identification of these organs and is summarized at the end of the lab manual.

Intralobular ducts are the

- intercalated ducts with low cuboidal epithel lining, and high cuboidal to columnar,
- striated ducts with with cuboidal to columnar epithel lining

Interlobular and interlobar ducts are the:

-excretory ducts cuboidal with transitions to pseudostratified columnar and stratified columnar

a. Mixture of serous and mucous acini with serous demilunes - diagnostic for submandibular gland. Serous cells have eosinophilic granules with round central or basal nuclei. Mucous cells are pale staining with flattened basal nuclei.
b. Predominately serous acini - diagnostic for parotid gland
c. Predominately mucous acini with serous demilunes - diagnostic for sublingual gland

104A Salivary gland composite, human, H&E
104B Salivary gland composite, human, Masson  

This slide gives you the opportunity to compare all three salivary glands on one slide. Pictures from this slide are shown above.
Notice that the sublingual gland that striated ducts are difficult to find. In some slides, you can see that the mucous acini form tubes and not strictly acini, hence the lack of intralobular ducts. The sublingual gland has a significant amount of serous demilunes.
The serous acini are much more prominent than they are in the submandibular gland.
The parotid gland has a significant amount of fatty tissue intermingled with the parenchymal cells so do not be fooled that this is a mixed serous/mucous gland.
The Masson stained slide does a much better job of contrasting serous and mucous secreting acini. The sublingual gland in Masson stain appears much more like a predominately mucous secreting gland than it does in H&E.

 
101A Parotid, MA
The parotid consists of serous secreting acini arranged in separate lobules all found together in a multilobular gland. The duct system consists of intercalated ducts, striated ducts, and excretory ducts. The intercalated ducts are the smaller ducts within each lobule, usually somewhat difficult to see. They have a low cuboidal epithelium and in this tissue, do not stain dramatically different from the parenchymal cells. The striated ducts are the intralobular ducts with the taller epithelium. Their color is a more intense blue color. The textbook describes striated ducts as having a columnar epithelium, but in this tissue they are cuboidal. They are numerous within each lobule. Excretory ducts are found in the septa separating the individual lobules. Their epithelium is taller and in the largest ducts, becomes stratified columnar. The secretory granules do not take up this stain so the apical cytoplasm of the secretory acini tends to look rather granular but without revealing secretory vesicles.

101B Parotid gland, human
This slide is probably 95% serous acini with 5% mucous acini and serous demilunes. The preservation is very good in this slide because cell boundaries are very clear. This should serve to remind you that there is no such thing as a purely serous or purely mucous salivary gland. You should keep in mind that there is always a chance of seeing serous secreting cells in mucous glands and vice versa. The diagnostic feature is the relative amounts In some areas of the slide, particularly around the edges, the serous granules stain up a very intense pink color. This is what you would expect of serous granules. Most of the other areas are relatively understained or faded. In a few places you can find, if you are perceptive, mucous acini surrounding a fairly large lumen. This is because the glandular epithelium is the duct itself near the end of the ascinus.
 

101C Parotid gland
This parotid gland presents a rather different picture than the previous one. Secretory granules can be easily seen in the ascinar cells. Although intercalated ducts can be found, they are the smallest of the ducts, the striated ducts are much more difficult to identify because their epithelium is almost stratified cuboidal. Another rather unusual feature of this tissue is the occasionally very large size of nuclei in the parencymal tissue. These nuclei are often 3-4x the size of the rest of the parenchymal cell nuclei. Excretory ducts can be found surrounded by dense connective tissue between lobules.

 
102B Submandibular = submaxillary Gland, Masson
As you scan this slide at 4x, your attention will be drawn to the system of ducts, but try and focus on the parenchymal cells and notice that there are pale staining inclusions within these regions.
These are mucous acini. Although there is variation in the shape of the nuclei in the serous secreting cells, in the mucous acini, virtually all of the nuclei are flattened and basal, a characteristic of mucous secreting acini. Some slides have streaks of tissue running through them. These are section wrinkles.
Intercalated ducts are relatively easy to see because they have a low cuboidal epithelium and appear relatively darker staining because of a relatively small amount of cytoplasm relative to the striated ducts.
Striated ducts have a clear cuboidal epithelium with a central to apical nucleus. They become more columnar as they approach the excretory ducts. Striations can be seen in the basal part of the cell, but it is sometimes difficult to convince yourself that you are seeing actual structure and not artifact. Try comparing the apical and basal parts of the cell.
Excretory ducts are found in the connective tissue and are mostly stratified columnar in this tissue. There are places where the excretory ducts appear pseudostratified because of the presence of occasional basal cells. This should serve to emphasize that there is usually a gradual transition from cuboidal to stratified columnar and that you should expect to see ducts of varying caliber and epithelial height. In each example of this slide there is an excellent cross section of a muscular artery with a clear internal elastic lamina, but no external elastic lamina.
There is also a ganglion. Although the ganglion is not diagnostic of a salivary gland, they are always fun to find (at least in my opinion). (Also, you should remember gross anatomy: the submandibular ganglion (parasymapathetic) is located very close to the submandibular gland.)

102A Submaxillary (submandibular) gland, human
You looked at this section back in the lab on epithelium.
Submaxillary (=submandibular) is a mixed serous/mucous secreting gland. The preservation here is quite good. Even terminal bars can be seen in the obliquely sectioned ducts. The basal striations in the striated ducts are very prominent. The nuclei of myoepithelial are present. The best way to identify them is to eliminate all the other more obvious structures. For example, the nuclei of the serous acini are round and stain moderately intensely with hematoxylin. The nuclei of the endothelial cells are dense staining and elongated. The mucous secreting cells have flattened, dark staining nuclei, which are likely to be the most easily confused with myoepithelial cells. After you have found all of those, what is left, except for lymphocytes are the myoepithelial cells. Their nuclei are dark staining but rather polygonal in appearance and quite variable in shape. This slide also has a considerable amount of adipose tissue sprinkled about it.
 

102C Submandibular gland, monkey
This gland has mixed serous and mucous secreting acini. The vast majority of the acini are serous secreting, which is typical of the human submandibular gland. Ducts of different types are very prominent and range from low cuboidal intercalated ducts to some stratified columnar ducts in the connective tissue septae.

103A Sublingual gland, human
This slide is more typical of sublingual. There is a high proportion of mucous acini while those serous secreting cells are mostly in demilunes. Myoepitheial cells are found throughout the salivary glands. However, they are sometimes difficult to detece around mucous acini becasue the ascinar cells have flattened basal nuclei also. A good way to find myoepithelial cells is to look for a dark staining nucleus between two ascinar cells. Complicating the location of myoepithelial cells is the fact that the capillaries also run between acini and the endothelial nuclei resemble the nuclei of the myoepithelial cells. Focus up and down through the tissue and this will help distinguish which feature is associated with a particular nucleus.

103C Salivary gland, sublingual, human
Virtually all of the acini are mucous secreting but a number of them have serous demilunes. The serous demilunes are not very dramatically stained relative to the mucous acini, but they are also distinguished by the presence of rounded central nuclei. The intercalated and striated ducts are hard to fine because they are normally not prominent as they are in submandibular and parotid glands.
 

 
1. Homogeneous glandular tissue with lobular organization (separated by C.T.)

5. Bile capillaries may be visible. These are identified as small round spaces centrally located on the boundaries of hepatocytes within the cords. Note that bile capillaries are formed by two hepatocyte’s cell membrane fastened together by maculae densae . Bile capillaries empty into bile ducts(Herring-canals) with cuboidal epithel lining at the margin of the helpatic lobule .

6.Dense, irregular connective tissue capsule (Glisson’s capsule) covered with mesothelium (i.e. a serosa)
 

105A Liver, pig, sec Compare with Fig. 17.2
105B Liver, pig
Pig liver has the same lobular organization as human liver except that the lobules are separated by distinct layers of connective tissue.
There are two types of slides here. 105a is Masson stained and 105b is sH&E stained. Both are equally good. In the Masson stained slide, a clear line of blue-green connective tissue separates the lobules. In addition, a thin blue line can be seen separating the hepatocytes from the sinusoids. This is the endothelial lining and the space of Disse. The bile canaliculi are not visible in this set of slides. Within the sinusoids are cells, clearly separated from the cords of hepatocytes with nuclei and pale staining cytoplasm. These are most likely Kupfer cells. Erythrocytes are present in the sinusoids but they appear as essentially ghosts without very little staining. In the H&E stained slide, the three features of the portal triad, hepatic artery, portal vein and bile duct are not easily seen everywhere but you can find some regions where they are easily identified. The connective tissue capsule is visible on one edge lined with a mesothelium. Notice the arrangement of hepatocytes and the large amount of capillary space in the organ.

106A Liver, human sec

There are 2 kinds of slides in this group, most of you have the newer slides. Some extremely large veins are present that are apparently sublobular or hepatic veins.
The space of Disse is enlarged somewhat artifactually, but the fact that it is visible should be utilized. The endothelial cell nuclei are displaced from proximity to the hepatocytes making them clearly identifiable.
Hepatocyte nuclei are round and pale staining with a prominent nucleolus. The endothelial cell nuclei are flattened, and in this particular tissue are in the sinusoids. The only other prominent cell in the parenchyma is the Kupfer cell and it seems to have an irregularly shaped nucleus that is fairly densely staining. However, Kupfer cells are easier seen in slide 109 below.

 
107 Glycogen, liver

This is perhaps the most informative of the liver slides, but since there are only 2 copies, it will be set up on the side shelf with one of the extra microscopes. See slide 111, which also contains liver with PAS stain. Glycogen granules stain a cherry red. There are a number columnar bile ducts as well as a sublobular vein, the latter identifiable by the large amount of CT surrounding it. Central veins have very little CT surrounding their lumen.

107 Rabbit liver and gall bladder, PAS&H
This slide has been stained with per-iodic acid-Schiff reagent to reveal glycogen granules in the liver which appear red. The hematoxylin stain has been added as a counter stain to reveal the individual cells. Notice that PAS stains very little anywhere else but in the liver. Notice that the glycogen granules are not uniformly distributed among the hepatocytes. They are more concentrated near the central veins and less concentrated near the portal triads. What might this indicate?

The gall bladder is not particularly well stained in this slide. However, you can identify the simple columnar epithelium and the smooth muscle. The serosa that surrounds both organs is preserved in only a few places.
 

109 Liver, phagocytosis (H5.73 Liver, sec. Rabbit, phagocytosis)

Just a few of you have this slide so share with a neighbor. This is an example of a tissue stained with the vital dye trypan blue. Trypan blue is a vital dye because it is taken up by live cells. The dye is injected into the living animal and macrophages phagocytize it. Everywhere in this slide that you see lots of dark (even black) granules is a macrophage and since this is a slide of liver, these macrophages are Kupffer cells. Some dye also appears to be taken up by the endothelial cells, suggested by the squamous distribution of the dye along the sinusoids. The reddish purple stain is provided to simply show some cellular detail. Some of these slides have a large hepatic vein in them.

Liver, lipid stained 
This slide was stained with a lipid dye (oil red or Kongo red) which brings out the distribution of fat in the liver .
Normally, adipocytes can be found only in the interstitium of the liver, forming clusters near the large veins.
However, several pathological conditions like severe obesity, prolonged fasting, A and E hypovitaminosis, viral infections (Hepatitis B &C), portal hyertension, toxin exposure, (alcohol, organic solvents) can induce accumulation of lipids in the hepatocytes themselves ("fatty liver"). Note that this is a reversible condition, after the removal of the toxic agent or return to proper diet promotes the hepatocytes’ natural ly excellent recovery.
 

Cirrhosis of the liver 
If the conditions mentioned at the previous slide are maintained for a long period of time, the pathological changes in liver structure become irreversible. It means loss of functional parenchyma (hepatocytes) for connertive tissue and irreparable changes in the lobular structure of the liver (cirrhosis).
Notice in this slide how fibrous connective tissue has obliterated much of the lobular structure of the liver. Some lobules remain, but the triads are located in inclusions, which appear not to be fed by hepatic arteries and veins.
The hepatocytes have different sizes depending on their distance within the remaining lobules.
Central veins are not easily visible. Notice also the higher concentration of cell nuclei toward the center of the remaining lobules. Despite the degradation in structure, the elements of the portal triads, hepatic artery, portal vein and bile duct can still be found in the fibrous tissue.

Hepatoma, primary cc. of the liver

The purpose of studying this is to compare normal hepatocytes and tumorously transformed liver cells. You will not be responsible for the pathological characterization of the primary liver carcinoma (cc.) or for its differential diagnosis from metastatic tumors of the liver in this course.
Cancer (carcinoma) is a malignant tumor of epithelial origin. In this case hepatocytes (derivatives of the endoderm lining foregut) lost the control over cell division (the ability to stop cell divisions when the daughter cell makes a contact with the neughbor= contact inhibition). Also, the hepatoma cells are less differentiated (more primitive-looking), more intensely dividing cells.
In this slide, the cancer cells are smaller with darker cytoplasm than the hepatocytes and have little if any organization. In some regions, you can see them invading the parenchyma, in other regions they appear to have completely obliterated the lobules. Some normal parenchymal structure remains.

  1. Lacks typical G.I. layering. Layering consists of

4. Muscularis externa: irregularly arranged layer of muscle.

 
Gall bladder sec. Compare with Fig. 17.12

This slide should be set up on the side counter because there is only one copy of it. This is the best gall bladder so if you have trouble with the others, you can examine this one. The only defect is that the smooth muscle is not highly stained but the smooth muscle can still be seen because the arrangement of nuclei in the connective tissue is very different on either side of it. The serosa is very evident on one side. The large blood vessels in the CT tissue are common features of gallbladder.

110C Gall bladder, human

This is perhaps the best gall bladder slide that we have more than one copy of even though the epithelium is only partially intact along the surface. Features to note: The epithelium consists of only a single cell type. Even though the epithelium is folded and reveals a villus type of structure, the absence of goblet cells in the epithelium should tip you off that this is not small intestine. The muscularis externa reveals several oriented bands of smooth muscle separated by CT. In small intestine, the muscularis externa would be confined to 2 well defined bands perpendicular to each other. Note also that there are no myenteric plexi between the smooth muscle bands. There is an intact serosa.

110B Gall Bladder, Masson, human

This is a pretty good gallbladder slide. The epithelium is reasonably intact and the layering is clear. There are some aspects that could be confusing and cause a misidentification as small intestine. The muscularis externa is pretty close to the epithelium and appears as several bands of muscle rather than a pair of discrete bands at 90° orientations as would occur in small intestine. Because the muscle is not so well confined, some of it appears like a muscularis mucosae. However, the muscularis mucosae in small intestine has two smooth muscle orientations the same as the externa and it is a generally continuous band, not the disrupted band that is seen in gallbladder. A serosa covers the outside connective tissue layer.

110A G. Bladder, H&E, human 
This slide has an unusual cut through the bladder. There is a natural edge on top and bottom side but in the middle, some slides show the surface epithelium cut in cross section revealing transverse cuts through apparent "villi". There is not a lot of epithelium left in the slide. However, where you find columnar epithelium, you can see that the muscularis is adjacent to the lamina propria. There is an extremely large amount of adventitia suggesting that this is a rather oblique section through the gallbladder wall. The brush border can actually be seen on the columnar epithelium in a few places, particularly where the epithelium has remained attached to the underlying basal lamina.
 
 

113A  Pancreas head shows the duodenal papilla.

112a and 112c have unusual stains but the preservation is not too good. The islets of Langerhans are difficult to see in 112A  and 112B and for that reason you might try and find them as an exercise. If you have trouble identifying islets in any of the slides, seek help. They are a key diagnostic of the pancreas and you need to be able to recognize them.

  1. Highly lobulated glandular tissue with delicate C.T. septa between lobules

a. Delicate, thin connective tissue capsule covered with a mesothelium on one surface (anterior)
b. Exocrine portion: closely packed serous secretory acini
c. Endocrine portion: islets of Langerhans

a. Secretory acini are both simple acinar and tubuloacinar
b. Acinar cells: basally located round nuclei with basophilic subnuclear cytoplasm (the ergastoplasm)
c. Zymogen granules (secretory granules) are acidophilic and located apically
d. There are no basket cells.

a. Centroacinar cells - squamous cells that are typically pale stained and located in the center of the acini. Centroacinar cells lead into intercalated ducts
b. Intercalated ducts: low, simple cuboidal epithelium. Lead into intralobular ducts.
c. Intralobular ducts: lined with a cuboidal epithelium. Located within lobules
d. Interlobular ducts: begin with low columnar epithelium which rises in height as luminal diameter increases. Located in C.T. septae. f. Main duct: tall simple columnar epithelium with occasional goblet cells. May have smooth muscle immediately under the epithelium
g. Striated ducts (typical of salivary glands) are absent

a. Cords of secretory cells (endocrine, secretum is taken up by capillaries)
b. Surrounded by a fine reticular capsule
c. Highly vascularized and lacking any excretory ducts. This is characteristic of endocrine tissue.
d. Poorly stained cytoplasm in H&E preparations
e. Presence is diagnostic for pancreas

 
112C Pancreas H&E, human 
In these slides, the ergastoplasm stains purple and the zymogen granules are red. You should have no problem finding islets in this tissue. Rather than clumps of cells (the acini), the endocrine cells of the islets are arranged in cords. This alone is sufficient to distinguish them from the acinar cells. However, in this preparation the islets are also rather pale staining. There is a large excretory duct in the middle of some slides. Centroacinar cells are not too difficult to find and are seen in about 10% of the acini. They have pale staining cytoplasm and the nucleus is not as dark as that of the acinar cells. Centroacinar cells are normally found at the vertex of the acini surrounded by the red stained zymogen granules.

113A tail of the pancreas 
This slide comes from the tail of the pancreas and is a rather typical pancreas slide. There is little difference between this slide and 128 so it is not necessary to look at both. Intercalated ducts are quite easy to find and look very much like Plate 87-2 in R, R&K. One slide has a muscular artery cut in longitudinal section along a rather long length, a somewhat unusual find.

114 Pancreas’ head, H&E
This section contains a piece of the duct of the duodenal papilla and some duodenum as well. The papilla has a rounded profile and is contained within the muscularis externa of the duodenum. A separate piece of duodenum is also contained in this slide, apparently not attached to the papilla region. The piece of attached pancreas is typical. The duodenum has the interesting feature that the epithelium has very few goblet cells, although the brush border is quite apparent.

113C Pancreas, human

This is a rather thick section that contains at least 2 if not 3 cell layers. This makes it somewhat difficult to view. However, the preservation is quite good, it is only the superposition that makes the slide difficult.

 
112A Pancreas,monkey, Mallory-Azan

For the most part, these slides (112a and 112b) are rather thick and overstained to be of much use. Because they are thick and overstained the islets are difficult to find. It, however, worth your time to make the attempt. Some slides are unusual in that there are several parasympathetic ganglia embedded in the parenchymal tissue. The ganglia are identifiable by their "blue color", not to be mistaken for the tunica adventita around the blood vessels and the CT around the ducts. Satellite cells surround the neurons. Look for large excretory ducts in the tissue also.

 
112B Pancreas Ald-Thio-Gömöri, human
The staining distinguishes the insulin-producing ß-cells located on the periphery of the of the islet of Langerhans. (Basic fuschin condensed with acataldehyde produces a water- insoluble violet dye. This dye specifically stains the ß-cells to garnet).
Unfortunately, overall preservation masks the attempt. Islets are difficult to find because everything is generally the same color. There is some tendency for the peripheral cells in the islets to stain darker than the internal cells but you may have a hard time convincing yourself of this. You should have some idea of the arrangement of cells in the islets, even if the light microscopic preservations do not bring the differences out.

113D Pancreas, human
There are lots of islets in this tissue as well as a large excretory duct.