Rabbit anti gfap

Rats were anesthetized and perfused through the ascending aorta with NS, followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer. Lumbar enlargement segments were then harvested, postfixed at 4 °C overnight, and dehydrated in 30% sucrose. Afterwards, 30-μm free-floating transverse sections were cut at −20 °C in a freezing microtome. Sections were collected in phosphate-buffered saline and double-labeled to investigate the co-expression of NR2B and neuronal, glial, or microglial markers. Specifically, spinal sections were incubated for 48 h at 4 °C in a mixture of mouse anti-NR2B (1:200; Abcam) and rabbit anti-PSD-95 (1:2000; Abcam), rabbit anti-NeuN (1:500; Abcam), rabbit anti-GFAP (1:500; Proteintech, IL), or rabbit anti-Iba-1 (1:1000; Abcam) antibodies. The sections were then incubated with a mixture of Cy3-conjugated anti-mouse IgG (1:200; Proteintech) and fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200; Proteintech), for 2 h at room temperature. Fluorescent signal was viewed with a fluorescence microscope (OLYMPUS, x-Cite 120, Japan) with the appropriate filters.

The morphology of microglia and astrocyte has been detected as previously reported [44 (link)]. The brains were harvested as described in HE and Nissl staining part, and immersed in 30% (w/v) sucrose at 4 °C until they sank to the bottom (n = 6 for each group). Serial coronal, 25 μm brain sections were prepared using a cryotome (Leica CM1800; Heidelberg, Germany) and then stored at –20 °C until use. During IHC, the following primary antibodies were used: rabbit anti-IBA-1 (1:500, cat# 019-19741, Wako, Osaka, Japan), a biomarker of microglia; polyclonal rabbit anti-GFAP (1:1000, cat# 16825-1-AP, Proteintech, Wu Han, China), a biomarker of astrocyte. The sections were incubated overnight at 4 °C and rewarmed at room temperature for 40 min. After being washed with 0.01 M PBS, the secondary antibody was added to the sections: Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:500, cat# 127803, Jackson Immuno Research Labs, West Grove, PA). After incubation with secondary antibodies for 3 h at room temperature, the sections were mounted onto glass slides. Digital images were obtained using a confocal laser-scanning microscope (LSM 800, Zeiss, Oberkochen, Germany).

Rats were perfused intracardially with PBS and subsequently fixed with 4% paraformaldehyde. After tissue was dehydrated and embedded in paraffin, a total of 5-μm section was cut and collected. Antigens were repaired via high temperature and pressure antigen repair using citrate buffer (pH=6). Tissue was cooled to room temperature and incubated in goat serum for 30 min, and incubated with the following primary antibodies at 4 overnight: rabbit anti-TH (1:500; Abcam), rabbit anti-IBA-1 (1:300; Abcam), rabbit anti-GFAP (1:500; Proteintech), rabbit anti-ΔFOS B (1:100; HuaAn Biotechology). For immunohistochemical staining, slides were incubated with biotinylated secondary antibody at 37°C for 15 min and visualized with avidin horseradish enzyme at 37°C for 20 min. Slices were visualized through DAB kit. For immunofluorescence staining, the second antibodies were anti-rabbit IgG Alexa Flour 488 (1:800; Abcam). Quantification of TH-positive neurons was performed through visually counting the number of TH-positive neuronal cell bodies blindly by two investigators. The images were recorded with a charge-coupled device camera and operated with the MetaMorph software. The results were obtained from the average. The mean value for the number of TH-positive neurons in substantia nigra was then deduced by averaging the counts of six sections for each rat.

The rats were anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg) and perfused with normal saline (NS), followed by 4% ice-cold paraformaldehyde in phosphate buffer. The lumbar 4–5 segments were removed, post-fixed, and dehydrated in 30% sucrose at 4°C. Next, 30-μm free-floating transverse cutting was performed using a freezing microtome at −20°C. After blocking with 10% goat serum for 2 h at room temperature to reduce non-specific binding, the sections were incubated for 48–72 h with the following primary antibodies: mouse anti-CXCR3 (1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Iba1 (1:400; Abcam, Cambridge, UK), rabbit anti-GFAP (1:500; Proteintech, Rosemont, IL, USA), and rabbit anti-NeuN (1:400, Cell Signaling Technology, Danvers, MA, USA). Subsequently, the sections were incubated with an appropriate secondary antibody (FITC-conjugated goat anti-rabbit or Cy3-conjugated goat anti-mouse, both 1:200 dilution; Beyotime, Shanghai, China) for 2 h at room temperature in the dark. Fluorescence signals were observed using a fluorescence microscope with appropriate filters.

WB is a commonly used method to evaluate the change of protein [45 (link)]. In present study, protein expression levels in CIP were detected by WB on POD 3 (n = 6 for each group). The supernatant of CIP tissue was collected as described in the ELISA part. The total protein levels were quantified by a bicinchoninic acid assay kit (Beyotime Biotechnology, Jiangsu, China). The following antibodies were used: rabbit anti-IBA-1 (1:500, cat# 019-19741, Wako, Osaka, Japan); polyclonal rabbit anti-GFAP (1:1000, cat# 16825-1-AP, Proteintech, Wu Han, China); polyclonal rabbit anti-Caspase 3 (1:3000, cat# 19677-1-AP, Proteintech, Wu Han, China); polyclonal rabbit anti-Bax (1:5000, cat# 50599-2-Ig, Proteintech, Wu Han, China); polyclonal rabbit anti-Bcl2 (1:1000, cat# ab59348, Abcam, United Kingdom); monoclonal mouse anti-β-actin (1:3000, cat# 7G6, CMCTAG, USA), and corresponding secondary antibodies. The blots were visualized using an enhanced chemiluminescence reagent (Millipore, MA, USA) and captured using ChemiDoc^TM MP System (Bio-Rad, UK). For quantification, the target protein levels were normalized to the β-actin levels and were expressed as relative fold changes. The densities of protein blots were analyzed using Image J Software by the researchers who were blind to the experimental design.