High-Resolution Imaging of Spinal Cord Neurons

To gain in confocal imaging resolution, we undertook STED microscopy imaging followed by deconvolution treatment on spinal cord microtome sections from a combination of Tg(UAS:TagRFP-CAAX;cmlc2:eGFP) and Tg(pkd2l1:Gal4) transgenic lines carrying the espin^icm26 mutation. The original IHC protocol [21 (link)] was adapted using the goat antirabbit IgG Alexa Fluor 594 secondary antibody (#A-11012; ThermoFisher) diluted to 1/200 to reveal the membrane-tagged TagRFP labeling in CSF-cNs in a STED-stable manner. The ZO-1 staining to outline the central canal was kept unmodified. Imaging of both ZO-1 and TagRFP-CAAX was carried out on a Leica TCS SP8 STED 3× confocal microscope using a 93× glycerol objective. ZO-1 labeling was excited with a white laser at 488 nm (3.5%) and detected on a HyD detector from 500 to 550 nm. TagRFP-CAAX labeling was excited with a white light laser at 590 nm (8.5%), depleted with a 775 nm depletion laser (80%), and detected on a HyD detector from 600 to 650 nm, gated from 0.6 to 5.37 ns. Images were then processed by deconvolution using Huygens software (SVI, the Netherlands). To assess the vertical extension of CSF-cN apical extensions in espin wild-type and mutant fish, we drew polygons outlining the apical extension and fitted an ellipse to extract the “minor” measurement using the polygon tool in Fiji.sc/ [68 (link)].