Af1056

Modifications and continuous updates to the protocol can be found at http://www.idisco.info. Pregnant females were euthanized with a rising gradient of CO2 and embryos were harvested through manual dissection. All harvested samples were post-fixed overnight at 4C in 4% PFA in PBS. Embryos were processed with the iDISCO+ immunolabeling protocol, as detailed previously (Renier et al., 2016 (link)). Embryos were stained with the following primary antibodies: cleaved caspase-3 (Cell Signaling 9661) at 1:500 and TrkA (R&D AF1056) at 1:500. Cleared iDISCO+ samples were imaged on a light-sheet microscope (Ultramicroscope II, LaVision Biotec) equipped with a sCMOS camera (Andor Neo) and a 2x/0.5 objective lens (MVPLAPO 2x) equipped with a 6 mm working distance dipping cap. Version v285 of the Inspector Microscope controller software was used. The samples were scanned with a step-size of 3 mm using the continuous light sheet scanning method with the included contrast adaptive algorithm for the 640 nm channel (20 acquisitions per plane), and without horizontal scanning for the 568nm channel.

Anti-TrkA antibody (R&D systems, AF1056, RRID:AB_2283049) was directed against mouse myeloma cell line NS0-derived recombinant rat TrkA Ala33-Pro418. The antibody shows approximately 5% cross reactivity with recombined human TrkA and less than 1% cross reactivity with recombined mouse TrkB and TrkC. On Western blot with rat striatum lysate the antibody recognizes a 140kDa band specific for TrkA. (manufacturers technical details). The labeling pattern in mouse TGG is in line with previous data, labeling nociceptive neurons (Matsumoto et al., 2012 (link))