Skip to main content
. 2019 Jun 19;39(25):4864–4873. doi: 10.1523/JNEUROSCI.3105-18.2019

Figure 1.

Figure 1.

Correlative light and electron microscopy approach to image the AIS. A, Top, Schematic representation of the CLEM approach depicting a side-view of the sample preparation; bottom, example images (top view). Rat hippocampal neurons were grown on pieces of Aclar film (Jiménez et al., 2006). Cells were labeled with anti-neurofascin, fixed, and labeled with fluorescent antibodies as well as 15 nm gold. Target neurons were imaged and the positions on the Aclar were marked using a needle. After further fixing and postfixing, Aclar pieces with the regions-of-interest (ROIs) were cut out and embedded into Epon resin. Then the Aclar pieces were removed, leaving the neurons and the needle markings upside down into the Epon block (Jiménez et al., 2010). The positions of the target neurons were further marked using a microdissection setup (Kolotuev et al., 2009). We drew 1 and 2 lines along the AIS to easily distinguish the left-right orientation and drew a 45° angled line pointing to the target neuron, so that while approaching the ROI, the distance between tow laser marks is the same as the distance remaining toward the ROI (see also B and C). To make these laser engravings visible during the trimming and sectioning of the Epon block as well as during the imaging by EM we applied a thin layer of gold using a sputter coater and wiped the excess away of the flat surface before covering the sample with a drop of Epon, leaving only the engravings filled with gold (visible in C). B, High-magnification of the neurons in Epon after marking the position using a laser dissection microscope. Red dashed lines represent the positions of the sections in C with the arrowheads pointing to the laser engravings seen in C. The small red line is the position of the EM image in D. C, 300 nanometer sections made while approaching the AIS to assess the position. Arrowheads point to the laser marks, and the neuronal cell bodies are indicated. D, EM image of a dendrite and the AIS from neuron II, where the AIS is decorated with 15 nm gold particles targeted to the extracellular part of the neurofascin membrane protein. Scale bars: A–C, 50 μm; D, 200 nm. CB, cell body.