Transmission Electron Microscopic Imaging of Synaptic Vesicle Endocytosis in Mouse Hippocampal Neurons

Protokol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

Electron Microscopy

1. Prepare a high K⁺ stimulation solution with HRP (horseradish peroxidase) as 31.5 mM NaCl, 2 mM CaCl₂, 90 mM KCl, 25 mM HEPES (pH 7.4), 30 mM glucose, 2 mM MgCl₂, 0.01 mM CNQX, 0.05 mM AP5, and 5 mg/mL HRP, then adjust to pH 7.4 with 5 M NaOH.
   1. Stimulate the hippocampal neuron culture with 1.5 mL high K⁺ stimulation solution at room temperature (referred to as K⁺) by addition of 1.5 mL to each well for 90 s. In the resting condition (referred to as R), apply the same concentration of 5 mg/mL HRP for 90 s, but with normal saline solution. For the recovery sample, apply high K⁺ stimulation solution as with the K⁺ sample, then rapidly wash and replace with normal saline and incubate for 10 min.
2. Fixation and staining
    
   1. Prepare 0.1 M Na cacodylate buffer using 21.4 g/L Na cacodylate at pH 7.4. Fix cells with 4% glutaraldehyde in 0.1 M Na cacodylate buffer for at least 1 h at room temperature. Wash three times with 0.1 M Na cacodylate buffer for 7 min each.
   2. Prepare Diaminobenzidine (DAB) solution, comprised of 0.5 mg/mL of DAB with 0.3% H₂O₂ in ddH₂O, and filter with a 0.22 µm filter. Apply 1.5 mL DAB solution for 30 min at 37 °C. Wash three times with 0.1 M Na cacodylate buffer for 7 min each.
       CAUTION: DAB is a toxic and suspected carcinogen. Please use gloves and lab coats.
      NOTE: Labeling with DAB occurs due to its oxidation by H₂O₂, as catalyzed by HRP. Small increases in the components result in an increased signal in the sample. Increasing the concentration of HRP speeds up the effect of the catalyst. Sufficiently large concentrations of H₂O₂ allow for debilitating side reactions with HRP, inhibiting the effect of labeling³². In this work, the concentrations of this labeling system were chosen based on currently available research.
   3. Incubate the neurons with 1.5 mL of 1% OsO₄ in 0.1 M Na cacodylate buffer for 1 h at 4 °C as post fixation. Wash three times with 1.5 mL of 0.1 M Na cacodylate buffer for 7 min each.
       CAUTION: Due to the toxicity and reactivity of OsO₄, keeping the sample on ice in a chemical hood is preferable in many cases to using a fridge for the incubation.
   4. Prepare 0.1 M sodium acetate buffer with 13.61 g/L sodium acetate and 11.43 mL/L glacial acetic acid at pH 5.0. Wash three times with 1.5 mL 0.1 M acetate buffer at pH 5.0 for 7 min each and incubate with 1.5 mL 1% uranyl acetate in 0.1 M acetate buffer at pH 5.0 for 1 h at 4 °C. Wash three times with 1.5 mL 0.1 M acetate buffer for 7 min each.
       
3. Epoxy embedding
    
   1. Dehydrate the neuron culture with single 1.5 mL washes of 50%, 70%, and 90% ethanol, for 7 min in each and then 3 washes of 1.5 mL 100% ethanol for 7 min each in a fume hood.
   2. Mix 485 mL/L bisphenol-A-(epichlorhydrin) epoxy resin, 160 mL/L dodecenyl succinic anhydride (DDSA), 340 mL/L Methyl-5-Norbornene-2,3-Dicarboxylic Anhydride (NMA), and 15 mL/L 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30) to create the epoxy resin. Mix thoroughly, then store under vacuum to remove air bubbles.
      NOTE: It is critical to remove air bubbles in the resin, especially those smaller than visible to the naked eye, because they can cause cavities in the resin during sectioning.
   3. Infiltrate the sample by replacing the ethanol with 50% epoxy resin in ethanol for 30 min at room temperature on a shaker, then 70% epoxy resin in ethanol for 30 min at room temperature on a shaker.
   4. Switch the epoxy resin solution with 100% epoxy resin and incubate for 10 min at 50 °C. Perform two exchanges of fresh 100% epoxy resin with incubations for 1 h at 50 °C. Add fresh 100% epoxy resin and allow to harden at 50 °C overnight and then at 60 °C for over 36 h to harden.
4. Remove each sample from the muti-well plate with a jeweler's handsaw. Select regions of interest, dense concentrations of cells, using an inverted light microscope, and then cut 70 to 80 nm blocks for sectioning by microtome. Mount the cut region in the microtome chuck and load the microtome. Position the chuck in the microtome and mount a diamond knife with the edge parallel to the surface of the block. Collect sections of 70 to 80 nm thickness directly onto individual grids.
5. Dissolve uranyl acetate into water for a 1% solution by weight, and separately dissolve lead citrate into water for a 3% solution by weight. Counterstain the sections by submersion with 1% aqueous uranyl acetate for 15 min and then 3% aqueous lead citrate for 5 min to improve the contrast of the samples.
6. EM imaging
   1. Examine the sections with a transmission electron microscope and record images with a CCD digital camera at a primary magnification of 10,000-20,000X.

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