The brains were homogenized on ice with a Ultrasonic crusher (HD3000, Wiggens, Beijing, China) in ice-cold lysis buffer (1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, RIPA) supplemented with protease inhibitors PMSF. Lysates were centrifuged at 10,000 × g for 20 min at 4 °C for two times, and the supernatants were collected. Protein concentrations were determined by a Bradford protein assay. Equal amounts of protein (30 μg) were separated by SDS-PAGE, and transferred onto a polyvinylidene fluoride filter (PVDF) membrane (Millipore). Membranes were blocked with 5% non-fat milk, and incubated at 4 °C overnight with anti-NG2, anti-GalC, anti-GDNF, anti-NT-3, anti-BDNF, anti-iNOS, anti-Arginase-1 and anti-β-actin (Cell signaling, Davers, MA, USA). Bands were visualized by HRP-conjugated secondary antibodies (Thermo Scientific, MA, USA) and chemiluminescence (ECL) kit under ECL system (Millipore).
Anti ng2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-NG2 is a laboratory reagent used for the detection and analysis of NG2 (neural/glial antigen 2), a proteoglycan found on the surface of certain cell types. It is used in various research applications involving cell biology, immunology, and neuroscience.
Automatically generated - may contain errors
Lab products found in correlation
Anti bdnf, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti arginase 1, by Cell Signaling Technology (1 mentions)
Anti inos, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl system, by Merck Group (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Isolectin b4, by Merck Group (1 mentions)
Anti gfap, by Thermo Fisher Scientific (1 mentions)
Anti sox2, by Abcam (1 mentions)
Anti tuj1, by BioLegend (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Anti doublecortin dcx, by Cell Signaling Technology (1 mentions)
Anti syn, by Abcam (1 mentions)
Anti pax6, by Abcam (1 mentions)
Anti neun, by Merck Group (1 mentions)
Anti map2, by Merck Group (1 mentions)
Anti mbp, by Merck Group (1 mentions)
Anti c myc, by Abcam (1 mentions)
3 protocols using anti ng2
1
Protein Expression Analysis of Brain Tissue
2
Retinal Vascular Pericyte Imaging
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Mice were anesthetized and perfused with PBS to eliminate the blood from the retinal vasculatures. Eyeballs were collected for trypsin digestion, and isolated retinal vasculatures were fully unfolded and dried on the slides, followed by post-fixation with 4% paraformaldehyde for 10 minutes. After the fixative was removed by rinsing, the vasculatures were blocked with PBS containing 5% normal goat serum and 0.3% Triton-X-100 for 1 hour. Subsequently, the vasculatures were incubated with anti-Epac1 (Cat. 4155S; Cell Signaling Technology, Danvers, MA, USA), anti-NG2 (Cat. AB5320; MilliporeSigma, Burlington, MA, USA), and isolectin B4 (Cat. I21412; Invitrogen, Waltham, MA, USA) at 4°C overnight. The following day, the vasculatures were rinsed and incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies at room temperature for 2 hours. Images were taken using confocal microscopy (LSM 800, Carl Zeiss Inc., Jena, Germany).
3
Immunocytochemical Characterization of Neural Stem Cells
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Immunocytochemistry was performed as described in Lee et al [22 ]. Briefly, differentiated NSCs were washed with D-PBS and fixed with 100% ice-cold methanol for 6 min. Fixed NSCs were permeabilized with 0.3% Triton-X 100/PBS for 30 min at room temperature and blocked with 10% normal goat serum (Vector, Burlingame, CA)/PBS for 1 h at room temperature. We utilized primary antibodies as NSC markers: anti-Pax6, anti-Sox2, and anti-c-Myc (Abcam, Cambridge, MA). Anti-Ki67 was used as a proliferation marker. For characterizing differentiation of immature neurons, brain-specific cell subtype markers were used, anti-Tuj1 (Biolegend, San Diego, CA) and anti-doublecortin (DCX) (Cell Signaling Technology). For mature neurons, anti-MAP2 (MilliporeSigma), anti-NeuN (MilliporeSigma), and anti-Syn (Abcam) were used, and for glial cell markers, anti-NG2 (Cell Signaling Technology, Danvers, MA), anti-Olig1 (MilliporeSigma), anti-MBP (MilliporeSigma), and anti-Gfap (ThermoFisher) were used.
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