Fig 1: AAV delivery via bilateral sciatic nerve direct immersion (SciNDi) in adult mice(A) Gross inspection of the left hindlimb musculature and exposed trunk of the sciatic nerve. Notice the iridescent (green) coloration, which is discreetly localized in the case of intramuscular (IM) injection, and is widespread following SciNDi. In the dissected views, sciatic nerve (ScN) is indicated by a yellow arrow head, while biceps femoris muscle (BF) is also marked. The insets show the un-dissected views and the area of the dissected muscle tissue is highlighted with a red oval.(B) qRT-PCR determination of vector genome copy numbers (AAV2-ITR/2N*ß-tubulin) comparing AAV delivery using bilateral IM injection or SciNDi.(C) qRT-PCR determination of eGFP (transgene) expression (??CT, relative to phosphate buffered saline (PBS) injected cohorts). Statistics in (C and D) (Mean ± s.e.m.; muscle, n = 6; sciatic nerve, n = 4; Mann-Whitney test, *= p = 0.05, ***= p = 0.005, ns = not significant).(D) Representative immunofluorescence (IF) images showing eGFP epifluorescence in the left L4 dorsal root ganglia following AAV delivery using bilateral IM injection (top panels) or SciNDi (bottom panels). Instances of eGFP positive cells are indicated by yellow arrows. Insets show 40× magnified views of eGFP alone (green) and eGFP with nuclear marker DAPI (blue). Antibody: eGFP (Cell Signaling Technologies #2555, Polyclonal rabbit, 1:500). High resolution IF images were acquired using a Zeiss observer inverted microscope equipped with colibri 7 LED illumination.(E and F) Dual-IF co-detection of eGFP and neurofilament light chain (NF-L) in the sciatic nerve following AAV delivery using bilateral IM injection (in E) or SciNDi (in F). Representative images (above: nerve trunk; below: 10× magnified views) showing eGFP alone (green), NF-L (red), merge of eGFP and NF-L and nuclear marker DAPI (blue). Antibodies: eGFP (Cell Signaling Technologies #2555, Polyclonal rabbit, 1:500) and NF-L (Cell Signaling Technologies #2835, mouse IgG1, 1:200). High resolution IF images were acquired using Olympus VS120 digital slide scanner equipped for brightfield scanning and fluorescence single-band emitters for Hoechst, FITC, Cy3, Cy5 and Cy7. See also Figure 3A (PBS). (G–I) Immunohistochemical (IHC) detection of eGFP in the left lumbar L4/L5 spinal ventral horn (VH-L) after IM application of PBS (in G), AAV delivery using IM injection (in H), or AAV delivery using SciNDi (in I). In H-I, select instances of eGFP immunostaining are highlighted in the cell bodies (red arrows: intense; blue arrows: clearly visible green arrows: faint/non-detected), and in intra-grey matter nerve cell processes (yellow stars). IHC was performed using an eGFP antibody (Cell Signaling Technologies #2555, Polyclonal rabbit, 1:500) and detected using DAB (3,3'-diaminobenzidine) chromogen. Haematoxylin was used as counterstain (20× magnified views, obtained using a Nikon brightfield microscope).