The overall goal of this experiment is to obtain reliable pre-embedding immunostaining of non-primate brain tissue for electron microscopy. This method can help answer key questions in the neuroanatomy field, such as the synaptic incidence of a given innervation in a precise brain structure. The main advantage of this technique is that it's an easy and low cost method to label specific innervations suitable for electron microscopy.
Begin by transferring 50 micron thick sections of acrolein fixed non-human primate brain containing the region of interest to a 12 well plate containing PBS. Wash the free floating sections three times in PBS for five minutes at room temperature, then incubate the sections in freshly prepared sodium borohydride solution for 30 minutes at room temperature. Rock gently without a cover.
Once the 30 minutes have elapsed, aspirate the sodium borohydride and add PBS to each well. Place the plate on a rocker and shake vigorously for 10 minutes. Repeat the wash two more times or until none of the reaction gas remains.
Block the sections by incubating in a solution of 2%normal serum and 0.5%cold fish gelatin diluted in PBS for one hour at room temperature with gentle rocking. Once the incubation time has elapsed, incubate the sections in primary antibody solution with gentle rocking overnight at room temperature. The next day, after washing the sections as before, incubate the sections in secondary antibody solution for one and a half hours at room temperature with gentle rocking.
After washing the sections as before, incubate in avidin-biotin-peroxidase or ABC solution for one hour with rocking. Next, prepare a fresh solution of 0.05%3, 3'Diaminobenzidine with 0.005%hydrogen peroxide diluted in TBS. After washing, incubate sections in the DAB solution for three to seven minutes with gentle rocking.
Once the brown precipitate has developed to the desired color, stop the reaction by quickly washing twice in cold TBS, then through a series of timed TBS and PB washes. Transfer the plate containing the DAB-stained sections to the venting hood. Transfer sections into a six well plate filled with PB and completely flatten the sections by carefully pipetting the PB out of the plate, then add osmium tetroxide solution drop by drop to avoid disturbing the flattened sections.
Cover the plate with aluminum foil to protect the sections from light and incubate for 30 minutes at room temperature without agitation. During the osmification, prepare water repellent epoxy resin by adding appropriate amounts of each component of the epoxy resin mix to a large plastic cup. Stir with a wooden stick or plastic pipette until a homogeneous brown color is obtained, then transfer equal quantities of resin to aluminum cups of appropriate size for the number of sections to be processed and allow it to rest.
Once the osmification period has elapsed, wash the sections three times in PB for 10 minutes with low speed rocking. After washing, dehydrate the sections in the following series of graded ethanol for two minutes each. Next, transfer the sections to glass vials filled with propylene oxide and incubate in propylene oxide three times for two minutes each to complete the dehydration process.
Then transfer the sections carefully, one by one, into the aluminum cups, avoiding contact with air as much as possible. Flat embed the sections in previously mixed water repellent epoxy resin and incubate them overnight under the venting hood at room temperature. The next day, coat glass slides and plastic cover slips with mineral oil, then soften the resin by incubating the aluminum cups at 60 degrees Celsius for 12 to 15 minutes at most.
Carefully flatten the sections on the greased side of the glass slide. Place the greased cover slip and carefully push out any remaining air. Incubate the slides at 60 degrees Celsius for 48 hours.
After 48 hours, remove the plastic cover slip, leaving the section encased in resin. Begin by using binoculars to locate the region of interest and then use a scalpel to cut a small quadrangular piece of tissue of approximately one milliliter squared. Next, file the tip of a resin block and then glue the quadrangular tissue piece onto it.
After drying, place the resin block in the ultramicrotome deck in the vertical position and use a sharp razor blade to gradually cut each side of the resin block to form a trapezoid with smooth sides. Next, put the deck in its horizontal position and rotate the block until the longest side of the trapezoid is facing downward. Place a diamond trimming tool or a glass knife in dedicated space.
Set the ultramicrotome to cut 300 micron sections at one millimeter per second. Adjust the knife to be vertically parallel to the quadrangular piece and to display a very small horizontal angle of approximately one degree and trim the surface of the quadrangular piece until the tissue starts to appear. Now, switch to an ultra 45 degree diamond knife equipped with a boat filled with distilled water to cut 80 micron thick sections.
Smoothen sections by going over them with a piece of absorbing paper tipped in xylene without touching the surface of the water, and collect serial sections on bare 150 mesh copper grids. Place the grids in a grid storage box then prepare a one to one solution of lead citrate stock solution and distilled water and filter through a 0.2 micron syringe filter. Cover the vial with aluminum foil to protect it from light.
Place each grid onto a drop of the diluted lead citrate solution, with the section in contact with the solution Then use small tweezers to hold the grid and thoroughly rinse it in two beakers containing distilled water. Gently remove the excess water with absorbing paper and place the grids in a grid box. After 30 minutes, the sections may be examined by transmission electron microscopy.
This electron micrograph of the squirrel monkey internal globus pallidus shows well-preserved material after acrolein-PFA transcardiac perfusion in the immunoperoxidase diaminobenzidine technique. As seen there, the procedure allows the myelin sheath of a large axon, indicated by the arrowhead, to be seen. This electron micrograph of the external globus pallidus demonstrates the dendritic profiles, indicated by the letter d, small unmyelinated axons, indicated by a, and axon varicosities, labeled av, can easily be identified.
The arrows show an example of an axon varicosity establishing a symmetrical synaptic contact with the dendritic profile. Immunolabeled elements can easily be identified by their cytoplasm or axoplasm filled with electron dense DAB precipitate. Here, a dendrite immunostained for choline acetyltransferase in the GPi receives a synaptic contact from an unlabeled axon varicosity.
This electron micrograph shows a myelinated axon in the GPe with a relatively intact myelin sheath whose axoplasm is immunolabeled for tyrosine hydroxylase. This example shows an axon varicosity in the GPi immunolabeled for serotonin transporter that establishes a symmetrical synaptic contact with a dendrite. In this example, the DAB precipitate lines the plasma membrane and the outer surface of organelles.
The axon varicosity shown here was observed in the GPe and immunolabeled for tyrosine hydroxylase. This represents an example of DAB precipitate entirely filling the axoplasm, with synaptic vesicles being visible but more difficult to delineate. Following this procedure, other methods, like pre-embedding double immunohistochemistry can be performed to answer additional questions like colocalizations of proteins or receptors at the synaptic level.
After watching this video, you should have a good understanding of how to prepare pre-embedding immunohistochemistry of non-primate brain tissue, proceed to the embedding in epoxy resin, and ultracut the region of interest into 80 micrometer thick sections suitable for electron microscopy.
