The overall goal of this procedure is to prepare mouse brain tissue for immuno electron microscopy. This is accomplished by first fixing the brain through profusion of the animal. The second step of the procedure is to cut the brain and process the sections for immunohistochemistry.
The third step of the procedure is to ossec, dehydrate and embed the sections. The final step of the procedure is to cut ultra thin sections. Ultimately, results can be obtained that show at high spatial resolution, the distribution of various proteins within different regions and cell types of the central nervous system.
Hi, I'm Maria Trombley from the Laboratory of Esca in the Department of Neurobiology and Anatomy at the University of Rochester. Today we'll show you a procedure for preparation of mouse brain tissue for immuno electron microscopy. We use this procedure in our laboratory to study interactions between microglia and synapsis during development, experience dependent plasticity and aging.
So let's get started. After fixing the mouse brain through intracardiac perfusion post, postfix it in approximately 10 milliliters of formaldehyde at four degrees Celsius for two hours. Wash the brain in four degrees Celsius, one XPB S3 times for 10 minutes each wash using a vibrator and ice cold one XPBS immediately slice the brain in transverse 50 micron sections.
Use a fine brush to transfer selected sections to a glass vial filled with four degrees Celsius. One XPBS store remaining sections in Cryoprotectant at minus 20 degrees Celsius for up to several years without alteration. Process a maximum of 10 freely floating sections for staining in each glass vial.
Use a transfer pipette to replace the one XPBS with fresh 0.1%Sodium boal hydride in PBS change solutions quickly to avoid letting the sections dry out. Incubate for 30 minutes, rinse the sections with one XPB S3 times for 10 minutes each. Taking care to remove all bubbles block and one XPBS containing 0.5%gelatin and 5%normal serum appropriate for the secondary antibody that will be used.
Incubate for two hours. Add the primary antibody diluted in blocking solution. Incubate for 48 hours.
Rinse with one XPB S3 times for 10 minutes. Each incubate with biotin conjugated secondary antibody diluted in blocking solution for two hours. Rinse three times 10 minutes in one XPBS as before.
Incubate with streptavidin horseradish peroxidase diluted in blocking solution for one hour. Rinse the sections one time in one XPBS and twice in one XTBS for 10 minutes each. To reveal the labeling incubate sections with a freshly prepared solution containing 0.05%dab and 0.01%hydrogen peroxide in one XTBS.
When the staining is visibly dark brown or the reaction has gone on five minutes, rinse the sections with one XTBS to stop the reaction. Then rinse once with one XTBS and twice with one x PB for 10 minutes each. In a fume hood, use a fine brush to transfer sections into a multi-well culture plate filled with one XPB.
Remove the PB from one well using a fine brush to spread the sections flat immediately. Add a solution of 1%osmium tetroxide in one x PB before moving on to the next, well incubate a room temperature for 30 minutes. Transfer OSM sections to glass vials.
Rinse with one XPB for three times 10 minutes. Dehydrate the sections in increasing concentrations of ethanol. Then propylene oxide as described in the text portion of this protocol.
Use a fine brush rinsed in propylene oxide to transfer the sections into freshly prepared dupant resin. For impregnation overnight, put the dish containing the sections into a 55 degree Celsius oven for 10 minutes. To soften the resin, use a brush rinse with propylene oxide to coat an chlor embedding film with a thin layer of resin.
Transfer the section onto the film and cover it with another embedding film evenly Distribute lightweights of about two grams on top to help the resin spread. Allow the resin to polymerize at 55 degrees Celsius for 48 to 72 hours in an incubator. After polymerization, remove the weights in the top layer of embedding film under a binocular microscope.
Select two by two millimeter areas of interest and use a razor blade to excise them from the film. Super Glue the areas of interest to the tips of resin blocks. Curing the 55 degree Celsius incubator for one hour.
Use a razor blade to trim the resin block to the shape of an isosceles trapezoid, the optimal shape for ultra thin suctioning. Using a microtome and a glass knife, remove the glue at the surface of the tissue to get a flat surface. For suctioning.
Fill the boat of the glass knife with double distilled water and cut a few sections between 0.5 and one micron. Use a perfect loop to transfer the sections on a super frost slide. Dry on an 80 degrees Celsius heating plate for one minute stain with 0.1%to luine blue for one minute.
Use a squirt bottle to rinse away excess stain with double distilled water Under a light microscope, examine the sections to distinguish the stain tissue from the resin. Cut semi thin sections until the border between tissue and resin approximately reaches the middle of the section. Replace the glass knife with a diamond knife and fill the boat with double distilled water.
Cut silver to silver gold ultra thin. Section 60 to 80 nanometers thick. Use fine inverted tweezers to carefully collect the sections on copper mesh grids.
Dry the grids on filter paper to enhance contrast. Use fine inverted tweezers to transfer a grid to a drop of lead citrate for two minutes. Delicately rinse the grid by plunging in three successive baths of double distilled water.
Dry the grid on filter paper store in a grid box for later examination on the electron microscope. Examine grids on the electron microscope to visualize the immunostaining, but also the morphology of cellular elements. They're intracellular organelles and their relationships with each other.
Here, representative immunostaining for IBA one is shown at the light microscopic level. The cellular distribution of IBA one is restricted to microglia, which confirms its specificity at the electron microscopic level. IBA one, immuno stained microglial peric carion and processes are visible.
The immunoperoxidase DAB staining appears as an electron dense precipitate. Other examples of IBA one, immuno stained microglial processes display different sizes and shapes. Higher magnification reveals various intracellular organelles inside one of the microglial processes and also the ultra structural relationships between another microglial process and nearby elements of neuro pill, including synapses.
We've just shown you how to prepare mouse brain tissue for immuno electron microscopy. When doing this procedure, it's important to remember to rigorously follow all the steps and manipulate the brain tissue delicately. So that's it.
Thanks for watching and good luck with your experiments.
