|
|
|
|
|
1999 Summer Scholars Program
|
Assessing the Specificity of APC CC-1 Labeling in vivo and in vitro
By Kristina N. Mays
Mentor: Dr. John W. McDonald, M.D. Ph. D.
Department of Neurology, Washington University School of Medicine, St. Louis, MO
|
|
Antibodies belong to the group of serum proteins known as
immunoglobins. Immunocytochemistry is a technique that utilizes the
highly specific antibody-antigen relationship to evaluate cellular
phenotype and mechanisms. Having a basic understanding of the scope,
or specificity, of each antibody allows scientists to effectively
employ them in lab. Adenomatous Polyposis Coli
is a gene found in tissues throughout the body. Mutations in the APC
gene result in a dominant allele based disorder called Familial
Adenomatous Polyposis (FAP), or Gardner's Syndrome. FAP is
characterized by cancerous tumors in the colon, thyroid, and brain as
well as the development of polyps in the upper gastrointestinal tract
and colon. The APC CC- I antibody is used with increasing frequency
in neuroscience laboratories because it has been experimentally
proven to label the cell bodies of oligodendrocytes in in vivo
studies.
Oligodendrocytes are components of the glia of the central nervous
system, the brain and spinal cord. The developmental cycle of
oligodendrocytes can be defined by a series of distinct proteins
expressed during certain phases of the cell's maturation. Using this
method, the oligodendrocyte lineage is divided into four stages: the
progenitor stage characterized by NG2 expression, the
preoligodendrocyte stage denoted by 04 expression, the mature
oligodendrocyte stage delineated by 01 expression, and the terminally
mature oligodendrocyte stage identified by myelin basic
protein expression. Mature oligodendrocytes, those with 01 and
MBP expression, are responsible for creating the myelin sheath, a
thin membrane surrounding neurons that allows for the rapid
transmission of signals between the brain and peripheral organs.
Damage to the white matter, myelin and surrounding cells, has been
studied in the mature nervous system. Analysis of the injury has
primarily been done through single labeling immunocytochemistry,
which employs one primary antibody and one secondary antibody. The
primary antibody labels the cell, and the fluorescent secondary
labels the primary, permitting the cell to be viewed under
epifluorescent light. APC CC- I has been used to label
oligodendrocytes in these studies because of its unique ability to
label only the cell bodies of oligodendrocytes. Other antibodies that
label oligodendrocytes tend to also label all processes and the
surrounding myelin, making the individual cells indistinguishable
when examining tissue from the damaged central nervous system. In
previous studies, it has been assumed that all cells labeled by APC
CC-I in the mature nervous system are myelinating oligodendrocytes
because there are few progenitors and preoligodendrocytes in the
mature nervous system; however, double labeling was never done to
prove this. After injury, there are inflated numbers of cells labeled
that researchers believe are mature oligodendrocytes; however, after
injury, mature oligodendrocytes can dedifferentiate and progenitors
produce new oligodendrocytes. Thus, this study sought to prove that
APC CC- I labels only mature oligodendrocytes by double-labeling
cultures of oligodendrocytes and cultures of embryonic stem cells
with a lineage-specific marker and APC CC-1. The following
observations were made:
|
|
in vitro
|
in vivo
|
|
|
Embryonic Stem Cell Culture
|
Oligodendrocyte Cell Culture
|
Spinal Cord Sections
|
|
NG2
|
light labeling
|
light labeling
|
|
|
04
|
|
|
|
|
01
|
|
|
|
|
MBP
|
dark labeling
|
dark labeling
|
strong labeling
|
|
GFAP
|
light labeling
|
light labeling
|
|
|
|
Note: Any labeling with GFAP is an indication
that the APC gene is expressed.
|
Based on this study, the APC CC-1 antibody labels mature
oligodendrocytes and lightly labels progenitors and astrocytes;
therefore, in analyzing white matter injury, double labeling with
NG2, GFAP and MBP is necessary to ensure that the damage is assessed
accurately.
This page was last updated on Fri, Jun 16, 2000 at 2:16:31 PM
by Tom Elgin with Userland Frontier.
