HCN1 and HCN2 immunostaining.

<p><b>A and B</b>: ABC immunostaining. <b>A:</b> with X100 objective, HCN1 and HCN2 ring staining (white arrows) is over the neuron perimeter and not in satellite cells (black arrows) thus probably membrane associated, (sn = satellite cell nucleus). <b>B:</b> in adjacent sections HCN1 and HCN2 show co-localisation, with some differences in intensity or location. Symbols in <b>B</b>: ‡ ring staining for both HCN1 and HCN2; x neither, o clear HCN1 ring but weaker HCN2; • clear HCN2 ring but weaker HCN1. <b>C–F:</b> Double immunofluorescence staining in L5 DRG neurons. <b>C and D</b>: HCN1 (C) or HCN2 (D) ring staining (red) are present in large, myelinated, neurofilament-rich (RT97 positive, green) neurons. There is also cytoplasmic HCN2 staining in a few RT97 negative, unmyelinated, small neurons (fine arrow in D). Symbols in C and D indicate examples of staining with: ‡ both antibodies, x neither, o clear HCN2 cytoplasmic but not neurofilament staining. <b>E and F:</b> Representative X100 images of HCN1 (E) and HCN2 (F) (in red) with Ankyrin G (AnkG, green) to indicate the nodes of Ranvier (arrows) in longitudinal sections of normal L5 dorsal root nerve. Yellow is indicative of co-localisation.