Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

The overall goal of this procedure is to grow primary rat neurons in such a way that their neurons are suitable for visualization by cryo-electron microscopy. This is accomplished by first preparing dishes with EM grids for plating primary neurons. The second step is to prepare and plate the primary neurons on EM grids.

Next, the vitrification of neurons on EM grids is performed. The final step is to collect images by cryo-electron microscopy, followed by post-processing and annotation. Ultimately, cryo-electron microscopy and tomography are used to show the ultra structure of neurites in a frozen hydrated state.

The main advantage of this technique over existing methods is that it can be used for 3D visualization of frozen hydrated neurites at nanometer resolution without the use of chemical fixatives. Therefore, facilitating the assessment of morphological characteristics that are closer to the native state. Begin this procedure by examining the integrity of holy carbon on the gold EM grids.

Using a light microscope at a magnification of at least 25 times, make sure the carbon holes are larger than 98%intact. For plating primary neurons use a bunsen burner to flame sterilize the EM grids and concurrently render them hydrophilic. Immediately transfer the grid with the carbon side up to the center of the glass.

Bottom dish, place one EM grid per dish and use only pre sterilized glass bottom dishes. Then use a light microscope to check the grid integrity whilst still keeping the EM grid inside the glass bottom dish in a tissue culture hood, apply 250 microliters of the appropriate coating substance to the central glass area of the Petri dish. Slowly and carefully, make sure the appropriate coating substance covers the entire E EM grid.

Afterward, cover the Petri dish with its lid and incubate the specimens for one hour at room temperature. Next, aspirate all the polyol lysine mixture from the dishes with a sterile pipette tip attached to the vacuum tube in the hood. Avoid direct contact with the E EM grid.

Subsequently, use an adjustable air displacement pipette to carefully apply 250 microliters of sterile PBS to fully cover the EM grid in the central glass area of each Petri dish. Then aspirate the PBS from each dish. Repeat the procedure three times after that.

Allow the dishes with EM grids to dry in the tissue culture hood for 15 minutes. Checking under the light microscope. Make sure that they're completely dry and no bubbles of moisture are in the grid.

Otherwise carefully aspirate next to the EM grid to eliminate this extra moisture, the coated grid should be used immediately for plating the neurons. In this procedure, calculate the appropriate volume of cells for each dish in order to achieve a concentration of 50, 000 cells per milliliter per dish. The maximum amount of application should be 250 microliters to fill only the central glass area, not the entire dish.

Next, incubate the dishes for 30 minutes in a CO2 incubator at 37 degrees Celsius to allow the cells to recover and adhere after 30 minutes slowly add 1.5 milliliters of the warmed cell media to each dish and avoid touching the EM grid for hippocampal neurons. Change the media the next day and then change half of the media every two days for 14 days. In this procedure, prepare the equipment and all materials for freezing and storing the gold EM grids at cryogenic temperature, which include a vitrification device with a humidity chamber, calcium free filter paper, a pair of long flat point tweezers, a pair of fine point specialized tweezers for the vitrification machine, a doer of liquid nitrogen, a coolant container, and the EM grid storage box.

Next turn on the vitrification device. Set the humidity to 100%and the temperature to 32 degrees celsius. In the option section, set the block time to zero seconds.

This allows for manual blotting through the side window of the humidity chamber. Then stack three calcium free filter papers. Cut the stack into 0.5 centimeter wide strips that are about two centimeters long after that, bend them at a 90 degree angle such that one section of the paper is 0.5 centimeters by 0.5 centimeters.

This section will be used for blotting the specimen. Remove the middle paper and place it on another calcium free filter paper until use. Next, use the specialized vitrification to tweezers to carefully pick the EM grid from the dish.

Note the side of the EM grid that the neurons are growing on as the position will matter for the next step. Then use the black sliding lock on the tweezers to securely lock the tweezers onto the EM grid. Now insert the tweezers into the vitrification machine such that the side of the EM grid on which the neurons are adhered faces to the left and away from the circular opening hole on the side of the vitrification machine.

Then retract the tweezers that hold the EM grid into the vitrification machine. Afterward, place the coolant container filled with adequate LN two and liquid ethane in the appropriate holder of the vitrification machine using the appropriate screen command of the vitrification machine. Raise the coolant chamber upward until it's flushed with the bottom of the humidity chamber with the flat point tweezers.

Grasp one edge of the filter paper such that the shorter side is perpendicular to the tweezers. This face will come into direct contact with the EM grid for blotting. Now, carefully insert the filter paper into the side hole of the humidity chamber stably Hold the paper against the EM grid for 10 seconds, discard the paper and immediately plunge.

Freeze the specimen in the liquid ethane using the vitrification machine's automation. Afterward, carefully transfer the frozen hydrated EM grid to one of the slots in the grid storage. This is a light microscope image of the central area of an EM grid at 10 times magnification on which rat primary neurons have been growing for two weeks.

Here is the zoomed in view of the aqua box where the neurons and their neurite projections are observed. This is an electron micrograph of a neurite projecting outward from the neuron body at 4K magnification. The blue box is viewed close up here where the neurites internal features are clearly visible at 20 K magnification.

This video shows a 3D reconstructed stack of micrographs of a neurite from the primary culture of rat hippocampal neurons. It is imaged using tomography and followed by the corresponding 3D color annotation. Here is another video showing a 3D reconstructed stack of images of a rat dorsal root ganglion axon collected using cryo-electron tomography followed by the corresponding 3D color annotation and shown here is the montage of four 2D cryo-EM images of a separate rat dorsal root ganglion axon taken at 20 K magnification.

After watching this video, you should have a good understanding of how to prepare primary neurons on electron microscopy grids in such a way that is suitable for visualizing their neurons in a frozen hydrated state using cryo-electron tomography.