Transcript immuno 2015 - hippocampus

Immunolabeling of neurons and glia in
hippocampus to determine the location and
relative abundance of neurons and glia in the
hippocampus
Chris Strang PhD
VIS 729/GBS 730 July 2015
Antibodies are proteins produced by the body to attack
foreign invaders... bacteria, viruses, pollen etc. Antibodies
are specific, binding tightly to a particular part of the foreign
material. The target is called an antigen, or an epitope.
Reporter molecules, such as peroxidase or fluorophores
can be used to tag the antibodies so that the size or
location of the protein can be visualized.
Immunohistochemistry can be used to determine the
location and expression patterns of proteins in tissue or
cells. It can also be used to determine the morphology
and location of specific cell types.
The hippocampus
Glial fibrillary acidic protein is an intermediate filament protein that is expressed by by
astrocytes, and ependymal cells of the central nervous system (CNS). Antibodies against
GFAP can be used to label astrocytes.
Neurofilaments serve as major elements of the neuronal cytoskeleton supporting the axon
cytoplasm. They are abundant components of the axon. Antibodies against
Neurofilament-M can be used to label axons and as a marker for neurons
Hoescht is a blue florescent dye that stains DNA and is used as a label for cell nucleii
Slides with 15 um
hippocampal sections
obtained from P11 mice
have been prepared by the
DeSilva Lab
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Name
Wash the slides three times for 10 minutes each time in 0.1M
Phosphate-Buffered Saline containing 0.1% triton (PBS), pH 7.2-7.4
Drip the excess fluid from the slides, wipe back of slide and edges
with a kimwipe (be careful not to touch the tissue).
Using the superfrost marker, put your name on the white end of the
slide
Draw a circle around the tissue sections with the PAP pen and place
in humidity chamber
Cover tissue sections with 5% donkey normal serum (DKNS) in in
0.1M PBS containing 0.3% triton. This is used to block non-specific
binding
Incubate for 1 hour at room temperature
Antibodies are Y shaped molecules that have binding sites for
specific antigens.
There are 5 classes of antibodies
(although each class has many variations).
Each is called an immunoglobulin
(abbreviated ‘Ig’) and each type is allocated
a code letter. For example immunoglobin
type G is represented by IgG.
IgG- composes 75% of our
immunoglobulin pool. IgG stimulates
phagocytic cells, activates the complement
system, binds neutrophils, and can
neutralize toxins. Most importantly, it is the
only antibody that can cross the placenta
and confer immunity on the fetus. IgG also
has a single binding site.
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The FAB region is the antigen
recognition domain (binds to the
epitope).
Hinge region
The Fc region is the effector region,
it interacts with the other
components of the immune system
Once bound to the invasive antigen, an
antibody can trigger a variety of defenses by
the immune system, which are designed to
destroy the foreign protein.
www.jdaross.cwc.net/ humoral_immunity.htm
The binding of antibody with
foreign protein is a little like
a key fitting in a lock, or two
pieces of a puzzle locking
together.
Binding is also flexible. The arms
of the FAB region can bend and
flex to bind to a specific epitope
www.dep.anl.gov/S3A/ S3A-about-antibodies.htm
A critical point: A reporter molecule, such as peroxidase or a
fluorescent label can be added directly to the Fc region of an
antibody made against a protein of interest, without affecting
specificity.
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The use of a primary antibody that is directly conjugated to a
reporter molecule to detect proteins in tissue is known as direct
immunohistochemistry.
Indirect immunohistochemistry uses a primary antibody, and a
secondary antibody. In this case the secondary antibody has the
tag. This allows for greater amplification of the signal.
How do you “make” antibodies and visualize the
antibody binding?
To make primary antibodies, the epitope of the
protein of interest is injected into host species
(e.g. mouse or rabbit), which recognizes the
protein as foreign and creates antibodies to the
antigen (i.e. rabbit anti-NF-M, or mouse antiGFAP).
Indirect IHC requires secondary antibodies. To
create secondary antibodies, serum from the host
species (i.e. rabbit or mouse) is injected into a
second species (donkey). The injected serum
causes an immune response and species specific
antibodies are created (i.e. donkey-anti-mouse;
donkey-anti-goat secondary antibodies).
Fluorescent dyes such as fluorescein, rhodamine,
or DAPI or other reporter molecules are
conjugated to the purified secondary antibodies to
allow visualization.
If a peroxidase molecules are conjugated to the secondary antibodies,
chemical reactions are used for visualization. Usually only one color can be
visualized at a time.
If fluorescent molecules are conjugated to the secondary antibodies, then
fluorescent light of specific wavelengths is used for visualization.
To visualize two different proteins in the same cell or tissue, antibodies
specific to different proteins can be made in different species (i.e. rabbit
and mouse). The corresponding secondaries are then made in the same
species (donkey anti-rabbit, donkey anti-mouse) and tagged with different
color fluorochromes.
GFAP
NF-M
ms-a- GFAP
rb-a-NF-M
Dky-a-ms (488/green)
Dky-a-rb (Rho/Red)
Hoescht nuclear dye (AMCA/blue)
Antibody specificity
How do you know that the antibodies are binding only to what you want
them to bind to? Use of matched concentrations of IgG or pre- absorption
of the primary antibodies are used as controls for primary specificity.
Secondary antibodies are made against all the IgG from the serum of a
particular species. Thus they can recognize/bind to any protein from that
species. However, if there are proteins in the tissue that you’re labeling
that are similar to that of primary host species, the secondary might bind to
those proteins, and give you a false positive.
The blocking step eliminates potential endogenous binding sites for the
secondary antibodies, leaving only the primary antibodies for the
secondary antibodies to bind to. Omitting the primary antibody is used a
control for non-specific binding of the secondary.
Fluorescence Imaging
The visible spectrum spans the range of
wavelengths from about 390nm to about 750nm.
Shorter wavelengths have the highest frequency
and the greatest energy
Fluorescence is a process by which
some molecules absorb light of a
specific wavelength and then, after a
very short period of time, emit light at
a longer wavelength.
There is energy loss to the
environment and the molecule
relaxes to the lowest excited singlet
state.
Then after a few nanoseconds, the
molecule relaxes to the ground state
and this causes emission of a photon,
i.e. fluorescence.
Remember: High frequency, short λ
light, has more energy than lower
frequency, longer λ light. The difference
between the excitation λ and the
emission λ is called the Stokes shift.
Time
The basic idea is to deliver light of a given λ to the sample and then to
separate the lower energy emitted light from the higher energy
excitation light.
√ Blocking step – 5% donkey normal serum, 1 hour RT
drip excess of slides and add 150 uL primary antibody cocktail to the slides
Primary antibodies – overnight 4ºC
Mouse anti-GFAP binds to Glial fibrillary acidic protein, a marker of astrocytes
Rabbit anti-Neurofilament-M recognizes medium chain neurofilaments and can
be used as a marker for neurons
Day-2:
Secondary antibodies - 1 hour RT
Donkey anti-mouse conjugated to Alexa 488 (green)
Donkey anti-rabbit conjugated to rhodamine (red)
Hoescht nuclear stain (blue)
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Sectioned tissue
Wash
Block
Incubate with primary antibodies
Wash
Incubate with secondary antibodies
Wash
Coverslip
Visualize
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Sectioned tissue
Wash
Block
Rinse
Incubate with primary antibodies
Wash
Incubate with secondary antibodies
Wash
Coverslip
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Visualize (Monday –Thurs during week 2)
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Things to include in your lab notebook
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Hypothesis/purpose: What is your hypothesis? That is, what question is
this experiment designed to answer? What do you predict will happen? Your
hypothesis should relate to the predicted results..
 Do you expect any cell types to contain both GFAP and NF-M?
 Do you expect there to be more neurons or more glia?
 How can you use the nuclear staining to help determine the location and
abundance of cells?
 Where to you exect glia to be located?
 Where do expect the neurons to be located
Methods: Please note that fluorescence immunohistochemistry is the
method. Include the protocol, information about the antibodies and their
concentrations, the microscope, filter sets, and image acquisition program,
as well as any programs used to process your images.
Results: Describe the pattern of immunoreactivity. You should print out
copies of your images to include in your notebook.
Things to include if you choose this as a lab report
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Introduction: background and hypothesis,
Methods: Summarize the protocol and equipment used
Results: Describe the pattern of immunoreactivity. You should include figures and a
written description.
 Where is the labeling? What do you see?
 Was there only one cell type or more than one cell type labeled with each
antibody?
 Where are the labeled cells in relation to one another?
Discussion: How well did the pattern of immunoreactivity fit your initial hypothesis?
 Speculate on how the patterns of immunoreactivity might correspond to functions
(formulate a second hypothesis based on your results)
 The tissue that you used was from a young mouse, do you think that the circuitry
might be the same or different in other species or at other ages??
 What experiment(s) might you try next to test your new hypothesis?