Alexa fluor 647 conjugated ctb

Three days prior to the commencement of DMSO (vehicle) or 1NMPP1 treatment, all rats were intrapleurally injected with Alexa-Fluor647 conjugated CTB (C34778, Thermo Fisher), with CTB previously shown to reliably label all PhMNs in rats [66 (link)]. The 3-day period allowed for adequate retrograde transport to all PhMNs prior to any treatment inhibiting TrkB signalling (Figure 1). Three days prior to terminal procedures (i.e., day 11 of treatment), all rats were intrapleurally injected with Alexa-Fluor 488 conjugated CTB (C22841, Thermo Fisher) (Figure 1).
At day 14, following anaesthesia, all rats were euthanized by transcardial exsanguination and perfused with heparinized saline before perfusion with 4% paraformaldehyde (PFA) in 0.1M phosphate-buffered saline (PBS, pH 7.4). The fixed cervical spinal cord was then excised, post-fixed in 4% PFA in PBS overnight, then immersed overnight in 25% sucrose in PBS, prior to cutting 70 μm longitudinal (horizontal) cryostat sections. Sections were placed on gelatin-coated slides and cover-slipped with DPX mounting media (Fluka, Sigma-Aldritch, St Louis, MO).