Ab8135

Total protein from the spinal cord tissue was purified using protein extraction reagents containing 1% protease and phosphatase inhibitors. The protein concentration of each sample was quantified with Carmassi Bradford reagents (Thermo). An equivalent amount of protein (60 µg) was separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Bio‐Rad). After blocking with 5% (w/v) non‐fat milk, the membranes were further incubated with primary antibody solutions overnight at 4°C. The following primary antibodies were including: TrkA (ab‐76291, Abcam, 1:5000), FGFR1 (ab‐58516, Abcam, 1:1000), P‐AKT (sc‐514032, Santa Cruz, 1:1000), AKT (sc‐81434, Santa Cruz, 1:1000), P‐ERK (sc‐16982, Santa Cruz, 1:1000), ERK (sc‐514302, Santa Cruz, 1:1000), Bcl‐2 (60178‐1‐Ig, proteintech, 1:3000), Bax (60267‐1‐Ig, proteintech, 1:2000), cleaved caspase‐3 (sc‐373730, Santa Cruz, 1:500), GAP43 (ab75810, Abcam, 1:10 000), GFAP (ab7260, Abcam, 1:2000), NF‐200 (ab8135, Abcam, 1:5000) and GAPDH (K200057M, Solarbio, 1:5000). After three washed with TBST, the membranes were incubated with a 1:10 000 dilution of horseradish peroxidase‐conjugated secondary antibodies for 60 minutes at room temperature. Finally, signals were visualized by Chemi DocXRS + Imaging System (Bio‐Rad). All experiments were repeated three times.

Animals were killed 24 weeks post surgery following electrophysiologic assessment via an intracardiac KCl injection. Nerves were explanted, subjected to a series of increasing sucrose washes, embedded in optimal cutting temperature medium, and frozen for sectioning. Nerve cross-sections of 10 µm thickness were cut via micro cryotome at the proximal and distal ends of the nerve grafts. Sections were incubated with a neurofilament heavy chain (axons; NF200) antibody (Abcam Ab8135; 1:400) overnight at 4 °C, washed, and incubated again with fluoromyelin red (myelin; Invitrogen F34652; 1:300) and the NF200 secondary antibody (Invitrogen A11008; AlexaFluor 488; 1:1000) for 1 h at room temperature in the dark. Sections were then coverslipped in an aqueous mounting media containing DAPI. The number of axons was quantified by using images taken at ×4 magnification (to capture the entire nerve cross-section in a single image) using a counting algorithm in Cell Profiler Software. Among groups, the distal location was selected for comparison using a Kruskal–Wallis test with Dunn’s post hoc analysis comparing PNM vs. Autograft and PNM vs. Conduit. All groups within each group, proximal and distal axon counts were compared using a Wilcoxon matched-pairs signed-rank test. For comparisons of PNM concentrations, all groups were compared with each other using the same approach.

Rat sciatic nerves were fixed in situ in 4% PFA for 10 min, dissected, embedded in O.C.T. Compound (Tissue Freezing Medium; Solarbio, Shanghai, China). Sciatic nerve cryosections (5-μm thick) were incubated with acetone for 10 min at − 20 °C, washed in PBS/0.1% Tween 20, blocked for 30 min at room temperature (RT) in blocking buffer (0.3% Triton X-100/10% goat serum/phosphate buffer saline ¼ PBS), and incubated with primary antibodies overnight at 4 °C in blocking buffer. Sections were then washed 3 times in blocking buffer, and sections were incubated with secondary antibodies for 1 h at RT in the dark. Sections were washed again, incubated with DAPI for 5 min at RT, washed and mounted in Citifluor (Agar Scientific).
The primary antibodies used for IF were as follows: neurofilament (1:1000, Abcam, ab8135), SCG10 (1:500, Abcam, ab115513), IBA1 (1:100, Abcam, ab178847), and CD68 (1:100, Abcam, ab125212). All secondary antibodies were also purchased from Abcam. Images were acquired using a Leica TCS SP-II confocal microscope.

Proteins were extracted from tissues using RIPA lysis buffer (Roche, Diagnostics, Mannheim, Germany). Then, the lysate was loaded into SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). After blocking with 5% milk at room temperature, the membranes were incubated with primary antibodies against BDNF (ab220679, 1:1000; Abcam Inc., Cambridge, UK), GAP43 (ab11136, 1:1000; Abcam Inc.), NF-200 (ab8135, 1:1000; Abcam Inc.) or GAPDH (ab9485, 1:3000; Abcam Inc.) at 4 °C overnight. Subsequently, the membranes were incubated in HRP‐conjugated secondary antibody, goat antirabbit to IgG (ab205718, 1: 20,000; Abcam Inc.) for 1 h at room temperature. The expression was tested with enhanced chemiluminescence (ECL, Millipore, Bredford, USA) and ImageJ (NIH, USA) was used for image densitometric analysis and GAPDH was used as the internal control. Each experiment was performed in triplicate.

Brain slices were fixed overnight in 4% paraformaldehyde (PFA) and then in 30% sucrose for 2 days at 4°C. Subsequently, the slices were blocked with 5% bovine serum albumin (BSA) for 1 h and permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS) for 15 min. Primary antibodies, diluted in a blocking buffer, were added to the slices and were incubated overnight at 4°C. The primary antibodies used in this experiment were anti-NF (1:50, ab8135, Abcam, USA) and anti-maltose binding protein (MBP) (1:200, ab40390, Abcam, USA). The slices were washed three times with PBS and labeled with a fluorescence-conjugated secondary antibody for 1 h at room temperature (Alexa Fluor 488 and 594, 1:1,000, Life Technologies). Nuclei were visualized by mounting with DAPI (28718-90-3; Sigma Aldrich, USA).
For ROS staining in mitochondria, the MitoSOX™ Red mitochondrial superoxide indicator (M36008, ThermoFisher, USA) was used to label ROS in mitochondria. The MitoSOX was diluted according to the manufacturer's instruction and incubated with the slices for 3 h. After an extensive wash with PBS, the slices were replenished with the indicated culture medium.