The overall goal of this labeling methodology is to measure synaptic vesicle endocytosis. This method can help answer key questions in the neuroscience field such as the dispersion of formed endocytic vesicles. The main advantage of this technique is the resolution during imaging and the visualization of changes by examining the complete structure of a cell.
Generally, individuals new to this method will struggle because they fail to remove tiny air bubbles while preparing the epoxy or dry out the sample during the ethanol washes. To begin this procedure, prepare a poly-d-lysine coated six-well plate by applying 1.5 milliliters of 0.01%sterile filtered poly-d-lysine solution to each well for one hour at room temperature, then washing it three times with sterilized water. Next, prepare a high potassium stimulation solution with horse radish peroxidase and adjust the solution to pH 7.4 with five molar sodium hydroxide.
After that, stimulate the hippocampal neuron culture by adding 1.5 milliliters of high potassium stimulation solution to each well at room temperature for 90 seconds. In this step, prepare a 0.1 molar sodium cacodylate buffer at pH 7.4. Fix the cells with 4%glutaraldehyde in 0.1 molar sodium cacodylate buffer for at least one hour at room temperature, then wash the cells three times with 0.1 molar sodium cacodylate buffer each time for seven minutes.
Following that, prepare DAB solution and filter it with a 0.22 micrometer filter. Next, apply 1.5 milliliters of DAB solution to the cells for 30 minutes at 37 degrees Celsius, then wash the cells three times with 0.1 molar sodium cacodylate buffer each time for seven minutes. As post-fixation, incubate the neurons with 1.5 milliliters of 1%osmium tetroxide in 0.1 molar sodium cacodylate buffer for one hour at four degrees Celsius.
Afterward, wash the neurons three times with 1.5 milliliters of 0.1 molar sodium cacodylate buffer each time for seven minutes. Subsequently, prepare 0.1 molar sodium acetate buffer with sodium acetate and glacial acetic acid. Wash the cells three times with 1.5 milliliters of 0.1 molar acetate buffer each time for seven minutes.
After that, incubate the cells with 1.5 milliliters of 1%uranyl acetate in 0.1 molar acetate buffer for one hour at four degrees Celsius, then wash the cells three times with 1.5 milliliters of 0.1 molar acetate buffer for seven minutes each time. In a fume hood, dehydrate the neuronal culture with single 1.5 milliliter washes with 50%70%and 90%ethanol for seven minutes each wash, followed by three washes with 1.5 milliliters of 100%ethanol for seven minutes each time. Next, create the epoxy resin and mix thoroughly and store it under vacuum to remove air bubbles.
Infiltrate the sample by replacing the ethanol with 50%epoxy resin in ethanol on a shaker for 30 minutes at room temperature, then 70%epoxy resin in ethanol for 30 minutes at room temperature on a shaker. Switch the epoxy resin solution with 100%epoxy resin and incubate for 10 minutes at 50 degrees Celsius. Perform two exchanges of fresh 100%epoxy resin, each exchange is incubated for one hour at 50 degrees Celsius.
Then add fresh 100%epoxy resin and allow it to harden at 50 degrees Celsius overnight and then at 60 degrees Celsius for over 36 hours. Afterward, remove each sample from the multi-well plate with a driller's handsaw. Using an inverted light microscope, select the regions of interest that have dense concentrations of cells, then cut the blocks of four by five by eight millimeters cube for sectioning.
Afterward, counterstain the sections by submersion with 1%aqueous uranyl acetate for 15 minutes and then 3%aqueous lead citrate for five minutes to improve the contrast of the samples. In this study, electron microscopy was performed in cultured neuron and the clathrin-coated vesicles were examined in comparison to the control samples which were incubated with five milligrams per milliliter of HRP for 90 seconds in the absence of stimulus. The clathrin-coated pits were observed at a probability of 0.05 per bouton in the absence of stimulus.
In these images, stimulation with high potassium induced bulk endosome and synaptic vesicles uptake were identified by HRP labeling. Synaptic vesicles were defined as vesicles with a diameter less than 50 nanometers and bulk endosomes were defined as having a diameter over 80 nanometers or with a cross-sectional area of more than an 80-nanometer vesicle. Induced bulk endosome uptake was decreased by recovery with normal saline.
Once mastered, this technique can be done in 8-1/2 hours with three additional days to harden the epoxy if it is performed properly. Following this procedure, other methods such as immunogold labeling can be performed in order to answer additional questions like distribution of a target protein at various points in stimulation recovery. After watching this video, you should have a good understanding of how to stabilize and stain samples in preparation for imaging by electron microscopy.
