Each neuron in the brain is connected with other neuronal cells by approximately 7, 000 synapses. Except that those synapses in the mammalian brain are chiefly located on the small protrusions of the membrane called dendritic spines. Plasticity of the synapses and dendritic spines and the like, such important brain functions as learning and memory processes.
It is important to note that only electron microscopy gives a full insight whether a dendritic spine has a postsynaptic input. Various techniques on dendritic spines imaging are available. However, serial block face scanning electron microscopy gives unique possibility to image large volume of tissue with a high resolution.
Serial block-based scanning electron microscopy technique requires a special protocol of sample preparation which involves strong contrasting with heavy metals. My primary fixation during perfusion allowed us to use the same brains for lightened electron microscopy. Mice were sacrificed and perfused with a mild primary fixative.
The brain was cut into halves and one hemisphere was post fixed with immunofluorescence dedicated fixative, cryoprotectant, sliced using a cryostat and processed for immunofluorescence studies. Where the other hemisphere was post fixed with electron microscopy fixative, sliced with the vibratome and prepared for electron microscopy studies. Brain slices for serial block-based scanning electron microscopy studies were contrasted, flat embedded in resin, then a CA1 region of the hippocampus was mounted to the pin and imaged with the serial block-based scanning electron microscope.
The part of the protocol highlighted in a yellow box was featured in the video. Take the tissue, which has been fixed and cut into 100 microbiotic slices and wash it with cold phosphate buffer five times for three minutes each. Prepare a one-to-one mixture of 4%osmium tetroxide and 3%potassium ferrocyanide.
The final product will turn brown. Immerse the samples in this mixture. Then place the samples on ice.
And from this stage onwards it's important to protect them from light. Incubate them with gentle shaking for one hour. Meanwhile, prepare the TCH solution.
Mix 10 milliliter of double distilled water and 0.1 gram of TCH. Place it into an oven set at 60 Celsius for one hour. It is important to swill the solution from time to time.
When ready, cool it to the room temperature. Now wash the samples with double distilled water five times for three minutes each and then immerse them in filtered TCH solution. The slices will turn black.
Incubate them for 20 minutes at room temperature. Wash the samples with double distilled water five times for three minutes each and incubate them with 2%osmium tetroxide for 30 minutes, this time at room temperature. Again, wash the samples with double distilled water five times for three minutes each and place them into filtered 1%or amyl acetate.
Incubate them at four degrees Celsius overnight. Next day, begin with D-aspartate preparation. Mix lead nitrate with aspartic acid solution pre-warmed to 60 degrees Celsius.
Place the mixture in a water bath set at 60 degrees Celsius and adjust the pH to 5.5 with one molar sodium hydroxide. Close the vial with lead aspartate and leave it in 60 degrees Celsius for 30 minutes. The solution should be clear.
If it turns cloudy it must be discarded and a new one needs to be prepared. In the meantime, wash the samples with the gas double distilled water five times for three minutes each and keep them in the oven at 60 degrees Celsius for 30 minutes. Immerse the samples in the freshly prepared lead aspartate solution and incubate them in the oven set at 60 degrees Celsius for 20 minutes.
Prepare epoxy resin. Weigh the ingredients and mix the resin well for at least 30 minutes before adding accelerator DMP 30. Prepare virus with graded dilution of ethanol.
Then take the samples out of the oven and wash them again with double distilled water five times for three minutes each. In the meantime, mix the resin with 100%ethanol in one-to-one proportion to obtain the 50%resin. Mix it well.
Following this, dehydrate the samples in each dilution of ethanol for five minutes. Remember the samples must never dry completely. Infiltrate the samples first in 50%resin for 30 minutes, next in 100%resin for one hour, and then again in a fresh 100%resin overnight.
The next day, place the samples in fresh 100%resin for one hour and then embed them between fluoropolymer embedding sheets. Prepare pieces of embedding sheet which have been de-greased with ethanol and put aside the glass slides which will be used as support. Place a piece of embedding sheet on a glass slide, put a drop of resin on it, then transfer the sample carefully with the use of wooden stick.
Cover it with the second piece. Try to avoid air bubbles. Be gentle as the tissue is very fragile.
Carefully place another glass slide on top of the embedding sheet piece and cure the sample in the oven at 70 degrees Celsius for at least 48 hours. Separate the layers and cut a piece of the embedded sample with a razor blade. Transfer it to paraffin.
This will minimize the danger of losing the sample due to electrostatics. Take an aluminum pin that has been de-greased with ethanol. Mix a conductive epoxy well and use a little amount of it to mount the sample to the pin.
Cure conductive epoxy at 70 degrees Celsius for 10 minutes. Trim each side of the sample block with the diamond knife and then polish the face of the block until the tissue is exposed. Ground the sample to the pin using conductive paint and cure it.
To minimize charging, the samples should be also spotted coated with a thin layer of gold or gold palladium. Place the pin with the sample into the chamber of the serial block-based scanning electron microscope. Align the sample to the knife and then close the chamber and set the parameters.
Collect a stack of images. Using the described method, images of mouse brain tissue were obtained. The large image of the hippocampus one region was taken and the imaging was set in the center of stratum radiatum.
A stack of images was acquired and the objects of interest, such as dendritic spines and synapses, were segmented. Good quality tissue morphology was obtained with clearly visible membranes and synaptic vesicles and well-preserved mitochondria. Immunofluorescent studies of PSD 95 antibody confirmed that mild primary fixation and traditional fixation with 4%PFA give comparable results.
Presented protocol enables acquisition of brain tissue images with the use of serial block-based scanning electron microscopy, high-quality tissue morphology, and 12 visible membranes facilitate 10 right segmentation and reconstruction. My primary fixation made it possible to use the same brains for analysis of PSD 95 immunofluorescence and electron microscopy imaging dendritic spines. Here we use PSE 9, 500 budding.
However, the other can be used as well.
