Journal: STAR Protocols

Article Title: Recombinant adeno-associated virus mediated gene delivery in the extracranial nervous system of adult mice by direct nerve immersion

doi: 10.1016/j.xpro.2022.101181

Figure Lengend Snippet: Systemic AAV dissemination, eGFP expression in Schwann cells and inflammation at the site of AAV delivery using SciNDi (A) Dual-IF detection of eGFP and neurofilament light chain (NF-L) in the sciatic nerve trunk following bilateral IM application of PBS (vehicle). Representative images showing eGFP alone (green), NF-L (red), the merge images of eGFP and NF-L, and nuclear marker DAPI (blue). IF procedures and image acquisition were performed as described under E and 2F (nerve trunk and 10× magnified views). (B) Immunohistochemical (IHC) detection of eGFP in the right lumbar L4/L5 spinal ventral horn (VH-R) or both dorsal horns (DH-R and DH-L) after AAV delivery using SciNDi. IHC procedures and image acquisition were performed as described under G–2I. (C) qRT-PCR determination of vector genome copy numbers (AAV2-ITR/2N∗β- tubulin) comparing AAV delivery via IM route or SciNDi (Mean ± s.e.m.; SC: lumbar spinal cord, n = 6; B: whole brain, n = 4–6; and L: liver, n = 6; One- way ANOVA; ns, not significant). (D) qRT-PCR determination of eGFP (transgene) expression (ΔΔCT, relative to PBS injected cohorts). Statistics in A-B (Mean ± s.e.m.; SC: lumbar spinal cord, n = 6; B: whole brain, n = 4–6; and L: liver, n = 6; Mann-Whitney test: ∗= p ≤ 0.05, ns = not significant). (E and F ) Dual-IF co-detection of eGFP in sciatic nerve along with either the Schwann cell marker (S100, in E), or monocyte/macrophage marker (CD68, in F) following bilateral IM application of PBS, and AAV delivery using IM injection or SciNDi. Representative images (20× views) showing eGFP alone (green), S100 alone (red) (in E) or CD68 alone (red) (in F), and the respective merge images along with the nuclear marker DAPI (blue). Antibodies: eGFP (abcam #13970ab, Polyclonal chicken, 1:200), S100 (DAKO #Z0311, rabbit polyclonal, 1:200), CD68 (Novus Biologicals #NBP2-33337, rat mAb FA-11, 1:200). High resolution IF images were acquired using a Zeiss observer inverted microscope equipped with colibri 7 LED illumination. (G–I) Dual-IF co-detection of eGFP and and monocyte/macrophage marker (CD68) in hindlimb femoris muscle following PBS application using SciNDi (in G), and AAV delivery using IM injection (in H) or SciNDi (in I). Representative images (20× view, merge with DAPI, IF procedures and image acquisition were as described under Figure 3 F).

Article Snippet: Antibodies: eGFP (abcam #13970ab, Polyclonal chicken, 1:200), S100 (DAKO #Z0311, rabbit polyclonal, 1:200), CD68 (Novus Biologicals #NBP2-33337, rat mAb FA-11, 1:200).

Techniques: Expressing, Marker, Immunohistochemical staining, Quantitative RT-PCR, Plasmid Preparation, Injection, MANN-WHITNEY, Inverted Microscopy

Journal: STAR Protocols

Article Title: Recombinant adeno-associated virus mediated gene delivery in the extracranial nervous system of adult mice by direct nerve immersion

doi: 10.1016/j.xpro.2022.101181

Figure Lengend Snippet:

Article Snippet: Antibodies: eGFP (abcam #13970ab, Polyclonal chicken, 1:200), S100 (DAKO #Z0311, rabbit polyclonal, 1:200), CD68 (Novus Biologicals #NBP2-33337, rat mAb FA-11, 1:200).

Techniques: Marker, Virus, Recombinant, Plasmid Preparation, Electron Microscopy, Ointment, Sterility, Saline, Transferring, Adhesive, Software