essner as a whole. In the skin epithelium,


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PMI 201 Exam I. Reader/Van Winkle

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1. In the lung epithelium, ciliated cells function to propel
mucus towards the pharnyx1 to clear pathogens, particulates,2
and mucus1,2 to facilitate normal, unrestricted breathing. Structurally,
these columnar cells1 are found throughout the human airway from the
proximal tracheobronchial epithelium to the distal alveolar compartment of the
lung,3 and thus the cells contribute to the removal of waste along
the entire airway. A key feature of ciliated cells is the cilia, the
functional unit of the cell. Cilia are located on the apical surface of the
cell and are active projections that propel mucus and mucus-dissolved
substances towards the pharynx.1

In the gut epithelium, enterocytes
function to transport nutrients in the small intestine through its
apical surface, into the cell, and out its basal surface.4,5 This
absorptive cell is the main functional unit of the gut epithelium.4,5
Structurally, enterocytes are the most common cell in the gut epithelium4,5
to provide absorption necessary in the small intestine. A key feature of
enterocytes is the microvilli. Located on the villi on the apical surface of
the enterocyte, microvilli increase the surface area, and thus the absorptive
ability, of the cell4 and organ as a whole.

In the skin epithelium, melanocytes
function to produce melanin for keratinocytes. These cells are
stimulated by keratinocytes to produce melanin to be stored in keratinocytes. These
proteins contribute to the tan appearance of skin. Structurally,
melanocytes are branching cells in the stratum basale layer of the epidermis, allowing
communication with basal keratinocytes to thus be able to produce and share
melanin to protect DNA from UV light.6 A key feature of
melanocytes is their ability to produce and also secrete, via melanosomes, melanin.6,7
Pheomelanin and eumelanin, types of melanin, protect the skin by scattering and
absorbing UV light, aiding in ion storage, playing a role in redox reaction,
and breaking down free radicals.7

2.         First, stain all the
samples of the three conditions with AB/PAS, which stains proteoglycans and is
positively correlated to mucin production. View and capture the samples under a
light microscope. The negative control sham-treated mouse samples will display
the baseline amount of stain, correlating to mucin produced normally. The experimental
OVA-treated mouse samples, if of the asthmatic phenotype, should display more
stain, indicating more mucin production, than the sham-treated conditions and
would appear more like the sample from the asthmatic human positive control
than the sham-treated mouse.

            Next,
stain new slides of each of the conditions with H&E dye. This stains cell
nuclei blue and cell cytoplasm and ECM pink. View and capture samples under a
light microscope. The negative control sham-treated mice samples indicate a
normal airway. Experimental OVA-treated mice samples, if asthmatic, may display
goblet cell metaplasia, more lymphocytes, eosinophils, mast cells, and/or
neutrophils in the airway epithelium, a thicker basement membrane, and a
thicker layer of smooth muscle compared to the sham-treated samples and should
appear more similar to the positive control human asthmatic sample with respect
to immune cell inundation, goblet cells metaplasia, and inflammation than sham-treated
samples.

Third, stain new slides
with DAPI for nucleus visualization then tag them with primary antibody
specific for E-cadherin. Incubate, then add secondary antibody tagged with a fluorophore,
like Alexa488 (excited at 488nm, emits at ~520nm), to visualize under a
fluorescent microscope. Add negative control conditions by taking 2 prepared slides
from the sham-treated mice. Do not add primary antibody to one to ensure the
secondary antibody does not non-specifically bind. Add a primary antibody that
is not specific towards any protein in the sample. These conditions should not
fluoresce. The sham-treated samples should fluoresce a baseline amount green,
correlating to the amount of E-cadherin found in mice normally. The OVA-treated
samples, if of the asthmatic phenotype, are expected to show less E-cadherin
than the normal mice and be closer to the positive control human asthmatic
sample than the sham-treated samples. The resulting information may be quantified
using ImageJ software, allowing for statistical significance to be found.  

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