Principle of Fluorescent Immunostaining

1
Characterization of Cell Spreading and Focal
Adhesion Formation in B16F10 Melanoma Cells
John Ganz1 and Karen H. Martin2 1Biology
Department, Class of 2009 Eckerd College, St.
Petersburg, Florida 2 The Mary Babb Randolph
Cancer Center and the Department of Neurobiology
and Anatomy, West Virginia University,
Morgantown, West Virginia
Principle of Fluorescent Immunostaining
Abstract
Four Color Stain
Time Course of Spreading (Continued)
Focal Adhesion Kinase (FAK) is a member of a
family of non-receptor protein tyrosine kinases
that regulate cell survival, migration and
proliferation. This kinase is often over
expressed in invasive cancer types such as
breast, prostate and skin cancers. FAK inhibitors
are a target for therapeutic intervention and are
currently in phase I clinical trials. Melanoma
cancer cells were used in this study because of
their highly invasive characteristics and
increased levels FAK. Indirect immunofluoresence
microscopy was used to visualize phosphorylated
FAK and paxillin in focal adhesions. Melanoma
cancer cells were then fixed on plates at
different time intervals so the maturing of focal
adhesions could be observed and then quantitated.

3 Hour
3 Hour
3 hours- Cell in oblong shape. Focal adhesion
thick and long
A target protein is injected into an organism
such as a mouse. The mouses immune system will
make antibodies to this protein. The antibodies
are the purified out of the mouse and will
become the primary antibody for the target
protein. Mouse antibodies can then be injected
into another organism such as a rabbit. The
rabbits immune system will recognize the foreign
proteins and make antibodies to the mouse
antibodies. The rabbit anti-mouse antibodies can
then be purified and will become the secondary
antibodies. The secondary antibodies will then
have a fluorescent dye attached. The secondary
protein that is rabbit anti-mouse will then work
on any mouse antibody. In this way the primary
antibody is selective to particular protein
whereas the secondary antibody is selective only
to the organism that made the primary antibody.
Time Course of Spreading In Melanoma Cells
Melanoma cells were plated on fibronectin-coated
glass coverslips for various time to monitor the
process of cell spreading. The cells were then
stained with primary antibodies for rabbit
anti-pY397 FAK (the activated form of FAK) and
mouse anti-Paxillin (a marker for focal
adhesions). Secondary antibodies tagged with
green AlexaFluor 488 (anti-rabbit) and red
AlexaFlour 546 (anti-mouse) were used to
visualize the primary antibodies.
Outlined areas represent Focal Adhesions
1/2 Hour
A fluorescent molecule is excited by a specific
wavelength of light. When a fluorescent
molecule is excited, it will emit a different,
lower-energy wavelength of light. It is essential
that the excitation and emission spectra do not
heavily overlap with another fluorescent
molecules excitation and emission spectra. When
overlapping occurs, one color will bleed through
into another color creating a false
colocalization. The Fluorescent Spectra Viewer
graph (below) illustrates the four fluorescent
dyes used in these experiments.
½ hour- cells small and mostly symmetrical. Small
immature focal adhesions
FAK overexpression is correlated with poor
prognosis in cancers. FAK regulates functions
that contribute to formation of tumors and cancer
metastasis. FAK inhibitors are important targets
for therapeutic intervention.
1 Hour
Focal adhesions were outlined and then measured
by the program Image J in effort to quanitate the
number and size difference between the 1.5 and 3
hour time intervals. Three sets of experiments
with 3 images per time interval per experiment
were quantitated. The raw data were averaged for
each interval. These data show that focal
adhesions become larger and fewer in number
between the 1.5 hour and 3 hour interval.
1 Hour- Cells start to spread symmetrically.
Numerous Focal Adhesion form.
Melanoma Cell Lines
According to the American Cancer Society, an
estimated 8,110 people will die from melanoma in
2007. The cells raised in culture and
subsequently photographed were B16 F10 murine
melanoma cells. The F10 indicates the cells were
collected from metastatic tumors and then
serially passaged through ten generations of
mice. Growing and harvesting metastatic cells
from ten generations of mice selected for the
most invasive cancer cells.
Future Applications
2 Hour
The overexpression of FAK is a well known
characteristic of highly invasive/ metastatic
cancers. The understanding and inhibition of FAK
could lead to powerful cancer treatments. Pfizer
currently is in Phase I trials with a series of
FAK inhibitors.
2 hours- cell become polarized. Focal adhesions
become consolidated