The liver has a distinctive structure that can be compared to a suburban neighborhood. It is divided into lobules that contain hepatocytes arranged in tubes like houses, with sinusoids acting like roads. Each lobule contains a central vein and portal triad supplying materials. Our experiment stained the liver using immunohistochemistry and toluidine blue. Immunohistochemistry stained connexins between hepatocytes, laminin around structures, and cell nuclei. Toluidine blue stained nucleic acids, polysaccharides, and distinguished liver features. We faced challenges with tissue damage and non-uniform staining. Primary biliary cirrhosis involves immune attack on bile ducts leading to inflammation, fibrosis, and eventually liver failure.

1. Ismail Eddafali
05/03/2014
The Liver
The liver has many different structures that are quite uniform in their
arrangement. The comparison can be made to a suburb where houses are aligned
uniformly one after another, with gas stations and convenience stores found in equal
distances from each other. The liver is divided into classic lobules, which are somewhat
hexagonal in shape. The lobule can be thought of as a neighborhood. The liver lobule is
structurally made up of the parenchymal cells called the hepatocytes. The hepatocytes are
aligned in tube like arrangement much like the houses of a suburb. The area containing
the hepatocytes also contains structures called sinusoids, which can be compared to roads
or yards of the suburban houses. The classic lobule contains a central vein at the center
and a portal triad consisting of a portal vein, hepatic artery, and bile duct at the six
vertices. These structures are comparable to the gas stations and convenience stores as
they supply certain materials for the function of the liver. Each classic lobule is divided
by connective tissue septa comparable to the main roads that divide neighborhoods of a
city.
Our experiment consisted of labeling the liver using two different staining
methods. The first method was an antibody immunohistochemistry stain. We decided to
stain connexins, laminin, and cell nuclei, as these features are prominent and distinct in
the liver. The connexin stain allows us to view the gap junctions between the hepatocytes
of the liver as shown in figure 1. We used indirect immunohistochemistry, which requires
the use of a primary antibody to bind our proteins and a secondary antibody that will be
fluorescent and bind our primary antibody. This method allows for a higher fluorescence

2. to be present in our tissue. To bind connexins, we used an anti-connexin antibody from a
rabbit as the primary antibody. To bind and fluorescently stain our primary antibody, we
used the FITC conjugate-goat anti-rabbit IgG antibody that stained fluorescent green. The
nuclei were stained with a DAPI stain that strongly binds adenine-thymine regions of
DNA. Laminin is a major protein found in the basal lamina which is a prominent
structure found in many cell types as a layer of the basement membrane of a cell. To bind
laminin, our primary antibody was anti-laminin from a mouse. To bind our primary
antibody, we used a secondary antibody, which was anti-mouse IgG that stained
fluorescent red. The structures we predicted to be stained include the hepatocytes, the
portal triad, the central vein, and all cell nuclei. After staining was complete we saw that
The connexin stain labeled gap junctions between hepatocytes as shown in figure 1. The
laminin stain labeled portal triads, central veins and connective tissue septa, a feature that
we had not predicted would be stained as shown in figure 1. Finally, DAPI stained all cell
nuclei present.
The second method was a toluidine blue stain that stained nucleic acids blue and
polysaccharides purple as it is a basic dye staining highly acidic tissue structures. This
method allowed for similar structure staining as the immunohistochemical method. The
stain was able to distinguish many features of the liver including the connective tissue
septa, the hepatocytes, portal triads, central veins, sinusoids, and miscellaneous
vasculature. Figure 2 illustrates the cords of hepatocytes arranged around the central vein
and the sinusoids that align in a similar fashion.
We faced many challenges in our journey that came about from every stage in the
process. Some of our tissue was damaged in the process of fixation and some of it was

3. because of the age of the tissue. Tissue damage was present as a result of sectioning, as
knife marks were prominent in some mounted slides. In addition, certain areas exhibited
folding over, thickness variation and freeze fracturing. In the washing process due to
friction forces, before staining was conducted, there was some tissue loss this was
especially present in our immunohistochemistry stained slides. In the immunofluorescent
method, the antibody stain for connexin showed a prominent background, which
interfered in distinguishing the gap junctions between hepatocytes. During the toluidine
staining process, certain tissue areas stained longer and thus darker than others, which
created a gradation along the tissue. The mounting process may have moved tissue
around and caused folding over. Moreover, the mounting allowed air bubbles to
accumulate and this caused distortion of the tissue under the microscope.
Primary biliary cirrhosis is a disease that affects the bile ducts of the liver and is
believed to be a chronic autoimmune disease. The cause of the disease is unknown but
research has found a link to an immunological response against components of the
mitochondria. The symptoms associated with the disease include jaundice, itchy skin,
enlarged spleen, edema around the abdomen, and hepatic encephalopathy. Histologically,
the area of the portal triad will become inflamed which is caused by the gradual
destruction of the bile ducts. The area becomes highly concentrated with lymphocytes
and macrophages and granulomata appear. In addition, fibrosis around the triad and a
proliferation of small bile ducts begins to develop. The septa of the liver will become
more pronounced. Eventually the damage caused will become irreversible and the disease
will lead to liver failure.

4. Figure 1. Immunochemistry stained image at 20x magnification showing liver
hepatocytes (H) with distinctive connexon (C) stained gap junctions. Connective tissue
septa (S) is also prominent outlining the hexagonal classic liver lobule with a possible
portal triad (PT) visible.
Figure 2. Toluidine
stained image at
20x magnification
showing twin
central veins (CV).
The sinusoids (S)
and hepatocytes
(H) align around
the central vein in
a semi-linear
fashion.