Cy3 conjugated anti gfap

Free floating 45 μm thick sagittal sections were cut using a Leica SM2010 R sliding microtome and transferred to sterile TBS for storage. Sections were gathered and placed sequentially into wells (∼4 per well). Sections were then randomly selected from each well to perform antibody staining using the primary antibodies ACU193 (0.2 μg/ml), Alexa Fluor^® 555-conjugated NU4 (0.92 μg/ml), Cy3-conjugated anti-GFAP (1:800, Sigma) and the secondary antibody Alexa Fluor^® 633 goat anti-human IgG (1:2000, Invitrogen). Floating slices were rinsed 3 × 10 min with TBS and blocked with blocking buffer (10% NGS with 0.3% Triton X-100 in TBS) for 60 min at RT. Slices were then incubated with the respective antibodies in blocking buffer overnight at 4°C with gentle rotation. Sections were washed 3 × 10 min in TBS and incubated with secondary antibody for 3 h at RT with orbital agitation in the dark. Secondary was prepared in blocking buffer diluted 10-fold with TBS. Sections were then washed 3 × 10 min in TBS, mounted using ProLong Diamond^® antifade mounting media with DAPI (Invitrogen) and 24 × 60 mm No.1.5 glass coverslips (Thermo Scientific). Z-stacks of the brain sections were collected at 10× or 100× on a Leica SP5 confocal microscope and analyzed with ImageJ.

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.