Embedding procedures for acute slices

Slices were embedded in a solidifying aqueous mounting medium (Fluoromount-G, Southern Biotech, Birmingham, AL) using three different approaches:

For the conventional embedding method, the slices were mounted on a standard glass slide without any spacer and a coverslip placed on top. Care was taken to mount the slices with the recorded neurons (i.e. their somata) closer the upper surface when placed on the glass slides. Finally, coverslips were sealed with nail polish.

To prevent shrinkage of the mounted slice, the conventional embedding procedure was modified by adding a 300 µm thick agar spacer between slide and coverslip (Booker et al. 2014; Degro et al. 2015). To produce these spacers, agar (4%; Formedium Ltd, Hunstanton, UK) was diluted in 0.1 M PB and heated up to 90 °C. After cooling down, a solid block was formed, which was cut to the size of the coverslips (24 × 24 mm) and sectioned at 300 µm on a vibratome. Finally, a round hole with a diameter of 8 mm was stamped into the middle of the spacers. The spacers were collected and stored in 0.1 M PB until usage to prevent them from drying out.

In the new embedding approach, the agar spacer was replaced by a 300 µm thick metal spacer (Fig. 1). The metal spacer was made of stainless steel and had rectangular form with measures also defined by the coverslips, but exceeding those by a few millimeters in all directions (42 × 26 mm) to enable a stable contact and sealing with nail polish. The spacer had a round hole with a diameter of 15 mm in their center. The spacers were sourced from a commercial provider for custom-made metal objects (wh Münzprüfer, Berlin, Germany). For the embedding, one coverslip was glued onto the metal spacer using cyanoacrylate (UHU, Bühl, Germany), serving as a bottom plate during the mounting. After mounting the slice in the aqueous mounting medium, a second coverslip was carefully placed on the top of the assembly, making sure that no air was trapped, and sealed with nail polish.

Embedding of acute slices with the metal spacer system for light microscopic analysis minimizes shrinkage and improves image quality. a Schematic representation of the embedding process using the metal spacer system. The 300 µm thick metal spacer (left) and a fixed brain slice are sandwiched between two glass coverslips with a solidifying aqueous mounting medium (middle). A custom-made adapter is required for imaging the embedded slices (right). b, c Orthogonal projections of a full confocal image stack of a subicular pyramidal neuron onto the xy- (top surface) and the yz-planes (front surface) embedded with a metal spacer (b) and following the conventional embedding approach without a spacer (c). Note the substantial shrinkage and the weak signal in the deeper parts of the slice after conventional embedding. d Projection of the confocal image stack of another subicular pyramidal neuron onto the xy-plane embedded with a metal spacer. e–h High-resolution images of a dendritic segment (rectangle in d) embedded first with the metal spacer (e, f) and after re-embedding following the conventional approach without a spacer (g, h) viewed in xy- (e, g) and in yz-projections (f, h). The image in panel h was compensated for shrinkage for better comparison with panel f. Note the reduced detail of the contours of the dendrite and the absence of some spines (arrows in h and g) in the yz-plane after conventional embedding