Anti nf 160

The following antibodies were used for confocal microscopy. Primary antibodies to anti-CD4-alexa fluor 660 conjugated (eBioScience), CD11c-FITC conjugated (eBioscience), anti-ZIKV EVU-302 (Kerafast), anti-GFAP-alexa 633 conjugated (eBioscience), anti-NF-160 (Sigma-Aldrich), and anti-Iba-1 (Wako Chemicals).

The animals were sacrificed 3 months after hydrogel implantation. They were then deeply anesthetized with an intraperitoneal injection of overdose pentobarbital and perfused with physiological saline, followed by 4% paraformaldehyde in 0.1M phosphate buffer. The spinal cord was left in the bone overnight, then removed and postfixed in the same fixative for at least 1 week.
A 4 cm-long segment of the spinal cord with the lesion site in the middle was dissected, and a series of 40 mm-thick longitudinal sections were collected. Hematoxylin–eosin staining was performed, using standard protocols, and the slides were specifically evaluated using an Axio Observer D1 microscope (Carl Zeiss Microimaging GmbH, Oberkochen, Germany). For immunohistochemical studies, the following primary antibodies and dilutions were used: Cy3-conjugated anti-GFAP (1:200; Sigma-Aldrich, Saint Louis, MO, USA) to identify astrocytes, anti-NF 160 (1:200; Sigma-Aldrich, Saint Louis, MO, USA) to identify neurofilaments, and RECA-1 (1:50; Abcam, Cambridge, UK) to identify endothelial cells of blood vessels. Alexa Fluor 594 goat anti–rabbit IgG (1:200; Invitrogen) and Cy3-conjugated anti-mouse IgM (1:100; Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies.

Following the electrophoresis, proteins running on an 8% SDS-PAGEs were transferred to the nitrocellulose membrane, using a Mini Trans-Blot® cell at 40 V and 0.1 A for 16 hours in 1x transfer buffer with 24 mM Tris, 192 mM glycine, 15% ethanol, and 0.1% SDS. Transfer and equal loading of the samples were confirmed using Ponceau-S staining. After being blocked with 5% fat-free milk in Tris-buffer saline/0.1% Tween 20 (TBS-T), the membranes were incubated in the primary antibody diluted in 2.5% fat-free milk in TBS-T at 4°C overnight. The primary antibodies used were antineurofilament 200 (NF200; 1 : 1,000, Sigma), anti-NF160 (1 : 2,500, Sigma), anti-NF68 (1 : 2,500, Sigma), and anti- GAPDH (1 : 10,000; Abcam Ltd., Cambridge, UK). Then, the membrane was rinsed with TBS-T and incubated for 2 hours at the room temperature in horseradish peroxidase- (HRP-) conjugated anti-mouse secondary antibody (Invitrogen) diluted in the 2.5% fat-free milk in TBS-T. The immune complexes were visualized using the enhanced chemiluminescence (ECL) method (GE Healthcare, Piscateway, NJ). The densitometric analysis of the bands in various samples was performed on an image analyzer.