Anti neurofilament

For immunohistological evaluation, femoral nerves were harvested 5 days and 2 weeks after surgery and fixated in 4% buffered formaldehyde (VWR, Radnor, PA, United States) overnight. Subsequently, nerves were washed (distilled water) and transferred into 30% sucrose in phosphate buffered saline (PBS). 25 μm thick longitudinal sections were cut using a cryostat and mounted on gelatine-coated glass slides.
In order to evaluate axonal reinnervation, sections were blocked with 5% skim milk and subsequently stained with anti-Neurofilament (rabbit monoclonal; Abcam, United Kingdom) overnight at 4°C. After washing with PBS, sections were incubated with secondary antibody (anti-rabbit Alexa Fluor 488 Life Technologies, MA, United States) mounted for microscopical evaluation using glycerol.
Cresyl violet staining was performed as follows: cryosections were rinsed in distilled water and stained with 1% w/v Cresyl Violet solution pH 4 (Sigma-Aldrich) for 5–10 min. Samples were mounted with DPX mounting media (Sigma-Aldrich).

The cochlear explants were immediately fixed overnight in 4% paraformaldehyde at 4 °C, permeabilized and blocked by 1% PBST mixed with 10% donkey serum for an hour, then incubated with primary antibodies, including anti-parvalbumin (1:500 dilution, Abcam, 32895), anti-myosin 7a (1:500; Proteus Biosciences, 25-6790), anti-cleaved caspase-3 (1:500; Cell Signaling Technology (CST), 9664s), anti-HMGB1 (1:500, ab18256), anti-RAGE (1:500, ab37647), anti-C-terminal binding protein (CtBP2, 1:1000, BD Transduction Laboratories, BD Biosciences), anti-SOX2 (1:1000, R&D Systems, Minneapolis), and anti-neurofilament (1:1000, Abcam). Cochleae were incubated for 2 h with secondary antibodies at 37 °C. And DAPI (Sigma Aldrich, D9542) labeled the nuclei. All the images were taken by confocal laser scanning (Leica Microsystems, Germany). Photoshop (Adobe Systems Inc. USA) was used to assemble, examine, and manipulate these images.

The following primary antibodies were employed: anti-VAMP1 (1:200, generated as described in [23 (link)]), anti-CXCR4 (Abcam, cat. Ab 124824, 1:400, Cambridge, UK), and anti-Neurofilament (Abcam, cat. Ab 4680, 1:800). The α-bungarotoxin (α-BTx) (cat. B35451, 1:200) and Alexa-conjugated secondary antibodies (1:200) were from Life Technologies. Unless otherwise stated, all other reagents were from Sigma. NUCC-390 was synthesized as previously described in [20 (link)].
The M. nigrocinctus venom was a pool of more than 50 specimens collected in the central and Pacific regions of Costa Rica and kept at the Serpentarium of Instituto Clodomiro Picado (University of Costa Rica). Once obtained, the venom was freeze-dried and stored at −20 °C.

The following primary antibodies were used at the indicated dilutions: anti-CXCR4 (Abcam, cat. Ab 124824, 1:400), anti-syntaxin (Synaptic System, cat. 110111.00, 1:200), anti-Neurofilament (Abcam, cat. Ab 4680, 1:800). α-bungarotoxin (α-BTx) (cat. B35451, 1:200) and secondary antibodies Alexa-conjugated (1:200) were from Life Technologies. Unless otherwise stated, all other reagents were from Sigma. NUCC-390 was synthesized as previously described [46 (link)]
Purified Taipoxin and Oxyuranus scutellatus total venom were from Venom Supplies (Tanunda, South Australia). We estimated that our batch of taipoxin has a mouse LD50 of about 2 μg/Kg and that the Taipan venom is about 8 μg/Kg closely similar to value reported in the literature [47 (link)].