Immunohistochemistry of Synaptic Proteins

Immunohistochemistry was carried out as described in detail previously (Singer et al., 2016 (link)). Antibodies against C-terminal-binding protein 2 (CtBP2)/RIBEYE (rabbit, diluted 1:750; ARP American Research Products, Inc.™, Waltham, MA, USA) to detect ribbons (Khimich et al., 2005 (link)), neurofilament 200 (NF-200, mouse, 1:3,000; Sigma-Adlrich, St. Louis, MO, USA), parvalbumin (PV, rabbit, diluted 1:2,000; Abcam, Cambridge, UK), Synaptobrevin 2 (mouse, diluted 1:200; Synaptic Systems, GmbH, Berlin, Germany), and desmin (mouse, diluted 1:100; Abcam, Cambridge, UK) were used. Primary antibodies were detected using appropriate Cy3 (1:1,500, Jackson Immuno Research Laboratories, West Grove, PA, USA) or Alexa488 (1:500, Invitrogen Molecular Probes, Paisley, UK) secondary antibodies.
All samples were viewed as previously described (Zampini et al., 2010 (link)) using an Olympus BX61 microscope (Olympus, Hamburg, Germany), equipped with epifluorescence illumination and analyzed with CellSens Dimension software (OSIS GmbH, Münster, Germany). To increase spatial resolution, the slices were imaged over a distance of 15 μm within an image stack along the z-axis (z-stack), followed by 3-dimensional deconvolution using CellSens Dimension's built-in algorithm.