Figure 2
Whole-mounted immunohistochemistry (IHC) of ChAT (A,D,E) and whole-mounted in situ hybridization (ISH) of chat and vacht (B,C) in synganglia of unfed Ixodes females. (A) The synganglion of I. scapularis stained with antibody against D. melanogaster ChAT. (B,C) ISH of chat (B) and vacht (C) using their representative anti-sense probes in the synganglion of I. ricinus. (D) Ventral side of the I. ricinus synganglion after ISH of chat followed by IHC on the same specimen. Note that the dark spots are the SGs innervating opistosomal neurons (OsSG) visualized in ISH as seen in the confocal image. (E) Detail of the ventral posterior part of the synganglion (ISH followed by IHC) highlighting the OsSG neurons (in dotted circles) and associated axons. For a more detailed view see Videos 1 and 2. (F) Schematic illustration of the Ixodes synganglion highlighting the observed cholinergic neurons and their axons. Arrowheads—axonal clusters likely belonging to the OsSG neurons; arrows—axons exiting the OsSG cells and entering the opistosomal nerves (OsN), asterisks—lateral pedal axons, PN1-4—pedal nerves 1–4, E—esophagus; white square in (A) shows the disrupted part of the synganglion that facilitated the diffusion of the antibody inside the tissue. For the nomenclature of detected neurons see the section Whole-mount immunohistochemistry in the Material and methods. Scale bars are 50 μm. For ISH negative controls see Supplementary Fig. S2.
