The overall goal of this procedure is to perform ImmunoGold labeling to determine the ultra structural localization of synaptic proteins in rat hippocampal CA one parametal neurons. This is accomplished by first removing the CA one region of the organotypic slice, trimming the top corner to remember the correct orientation and incubating it in an osmium free fixative on ice. The second step is to dehydrate the fixed tissue and allow it to harden by infiltrating it with resin.
Next ultra thin sections are cut from tissues embedded in resin, and they are placed onto nickel grids. The final step is to perform immunohistochemistry on the section tissues. Ultimately, immuno electron microscopy is used to determine the localization of proteins in the synapse.Okay.
This method can help answer key questions in the synaptic plasticity field, such as how localizations of proteins within dendritic spines can help understand their role in synaptic function. Although this method is optimized for organotypic hippocampus slices, it can be also tremendously valuable for other neuronal preparations, including acute brain slices and primary neuronal cultures. Begin this procedure by placing the organotypic hippocampal slice into a polystyrene Petri dish containing fresh ice cold sorensen's phosphate buffer.
Add an additional one to two milliliters of ice cold buffer directly on top of the slices. In order to isolate the CA one's subfield of the slice, use a disposable scalpel to gently cut across the slice next to the dentate gyrus, which is parallel to the CA one cell layer. Then cut the remaining slice vertically to remove the CA three subfield and the sulu.
Next, cut a corner of the CA one slice to help identify the top surface of the tissue and carefully remove the tissue from the membrane using the backside of the scalpel. Gently transfer the tissue to a 12 well plate containing ice cold sorensen's buffer. Remove the buffer and add 0.5 to one milliliter of ice cold fixative at pH 7.3 diluted in sorensen's buffer so that it adequately covers the sample.
Then incubate for two hours at four degrees Celsius. Following incubation, remove the fixative from the well and wash the samples three times for 20 minutes in ice cold sorensen's buffer. Then incubate the sample in 1%Tannic acid weight per volume in 0.1 molar malate buffer at a pH of 6.0.
After 40 minutes, rinse away the tannic acid by washing the sample twice for 20 minutes in the malate buffer. Next, incubate the sample for 40 minutes in 1%Urinal acetate in malate buffer urinal acetate is sensitive to light and is radioactive. So handle with care and place the sample in the dark at four degrees Celsius during incubation.
Following the incubation, rinse the samples twice for 20 minutes each in ice cold malate buffer, then incubate for 20 minutes in 0.5%platinum chloride in inmate buffer. Finally, rinse away any residual platinum chloride by washing twice for 20 minutes. Each inmate buffer store the samples in malate buffer at four degrees Celsius until they are ready for further processing.
To begin immunohistochemistry, the fixed CA one regions are first dehydrated resin embedded and cut to obtain 60 nanometer ultra thin sections on nickel grids as described in the text protocol for this video. Next place a drop of 1%tween 20 phosphate buffer at pH 7.5 on a piece of clean paraform. Then gently pick up the grid with clean forceps and float it on the buffer with the section down incubate like this for 10 minutes at room temperature following incubation, drain excess liquid from the grid by placing section side up onto filter paper.
Also, remove any solution trapped between the arms of the EM forceps by gently wicking with a sliver of filter paper while ensuring the sections do not dry out completely. Then place a drop of 50 millimolar glycine onto the para film and float the grid section side down on the glycine solution at room temperature. While the sample incubates for 15 minutes, add 2.5%serum from the animal of the secondary antibody to a 2.5%solution of BSA and place a drop of this mixture onto the para film.
After the 15 minute incubation, dry the grid with filter paper and float on the blocking solution for 30 minutes. At room temperature, prepare the incubation chamber by placing rolled up Kim wipes along the inside of a clean plastic petri dish. Place a piece of clean param at the center of the Petri dish and then add filtered water to the Kim wipes.
Next place a drop of the primary antibody solution on the paraform and float the sample on it at room temperature or overnight At four degrees Celsius, the optimal time and temperature of incubation as well as antibody concentration need to be experimentally determined. After incubation with the primary antibody, wash the grid three times for two minutes each with 1%tween 20 phosphate buffer. Then incubate with secondary antibody using one to 20 anti rabbit or anti-US, coupled to 10 nanometer gold after one hour.
At room temperature, wash three times for two minutes each with 1%tween 20 phosphate buffer. Then postfix the sample in buffered 2%glutaraldehyde for five minutes. Remove excess gluar aldehyde by rinsing three times with 1%tween, 20 phosphate buffer, and then three times with filtered water rinsing for two minutes each time.
Once rinsed, incubate the grid in 2%urinal acetate for 10 minutes. During incubation, prepare a CO2 free chamber by placing a piece of paraform in the center of a glass petri dish. Then place 10 to 15 pellets of sodium hydroxide in the dish around the paraform to absorb CO2 in the air.
Keep the top closed for a few minutes. Next, use a pasture pipette and quickly transfer a small volume of freshly prepared Reynolds lead citrate solution to the paraform. Open the top just enough to insert the pasture pipette to minimize the reintroduction of CO2 into the chamber.
In three small beakers, prepare warm freshly boiled deionized water wash off the urinal acetate by dipping the grid in the first beak and gently swirl it around for 30 seconds. Repeat this step in the other two beakers. Once rinsed, open the top of the glass Petri dish just enough to place the grid on the drop of Reynolds lead citrate solution.
If possible, wear a mask while doing this to avoid breathing on lead citrate and to prevent formation of a precipitate. After 10 minutes, open the top slightly and remove the grid. Wash it three times by dipping it sequentially in three fresh beakers of warm distilled water.
Then let the grid dry on a piece of filter paper section up. The sample is now ready for viewing with the electron microscope ultra thin tissue sections of the ca one region of the hippocampus shown here as a transmission electron micrograph contain easily distinguishable dendrites and spines of parametal neurons. Identification of asymmetrical synapses between axonal terminal designated with a T and dendritic spines designated with an S is facilitated by the present of post-synaptic density and a well-defined plasma membrane.
The distribution of neuro Grandin in dendritic spines can be seen with the help of ImmunoGold EM labeling and is emphasized in these TEM images by the arrows. The radial distance of neuro grandin from the center of the spine to the plasma membrane of the spine can be measured as the normalized radial distance. A distance of zero corresponds to a particle lying on the membrane, and a distance value of one represents a particle at the center of the spine.
The distance of neuro grandin to the dendritic spine can then be shown graphically. Here its distribution as a normalized radial distance is shown in blue, and compared to a random distribution shown in pink, this graph shows neuro Grandin to be located closely to the plasma membrane. This technique paved the way for researchers who study the nervous system to explore localization and distributions of receptors receptor interacting proteins, transporters, and ion channels, and many others in different brain tissues, including the cortex, thalamus, olfactory bulb, and cerebellum.
Don't forget that working with fixatives can be extremely hazardous and the precautions such as wearing gloves and a lab coat and handling the fixatives in the film hood should always be taken while performing this procedure.
