1. Immunolabeling of neurons and glia in hippocampus to determine the location and relative abundance of neurons and glia in the hippocampus Chris Strang PhD July 2015 VIS 729/GBS 730 July 2015

2. 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. 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.

3. The hippocampus

4. 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

5. Name 1. 1. 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 2. 2. Drip the excess fluid from the slides, wipe back of slide and edges with a kimwipe (be careful not to touch the tissue). 3. 3. Using the superfrost marker, put your name on the white end of the slide 4. 4. Draw a circle around the tissue sections with the PAP pen and place in humidity chamber 5. 5. 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 6. 6. Incubate for 1 hour at room temperature Name Slides with 15 um hippocampal sections obtained from P11 mice have been prepared by the DeSilva Lab

6. Antibodies are Y shaped molecules that have binding sites for specific antigens. www.jdaross.cwc.net/ humoral_immunity.htm 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. 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. FAB region is the antigen recognition domain (binds to the epitope). The FAB region is the antigen recognition domain (binds to the epitope). Hinge region Hinge region Fc region is the effector region, it interacts with the other components of the immune system The Fc region is the effector region, it interacts with the other components of the immune system

7. www.dep.anl.gov/S3A/ S3A-about-antibodies.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

8. 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.Y YY 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.

9. 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 anti- GFAP). 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. How do you “make” antibodies and visualize the antibody binding?

10. 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. GFAPms-a- GFAP Dky-a-ms (488/green) NF-Mrb-a-NF-M Dky-a-rb (Rho/Red) Hoescht nuclear dye (AMCA/blue) 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.

11. 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.

15. 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

16. 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 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.

17. 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.

19. √ 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)

20. Sectioned tissue Sectioned tissue Wash Wash Block Block Incubate with primary antibodies Incubate with primary antibodies Wash Wash Incubate with secondary antibodies Incubate with secondary antibodies Wash Wash Coverslip Coverslip Visualize Visualize

21. Sectioned tissue Sectioned tissue Wash Wash Block Block Rinse Rinse Incubate with primary antibodies Incubate with primary antibodies Wash Wash Incubate with secondary antibodies Incubate with secondary antibodies Wash Wash Coverslip Coverslip Visualize Visualize (Monday –Thurs during week 2)

22. Things to include in your lab notebook 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.

23. Things to include if you choose this as a lab report 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?