At the age of 8 weeks (1 week after the exosome injection), the mice were sacrificed by decapitation, and the brains were immediately removed. Homogenates of each hemisphere were separately and serially processed for Western blotting and RNA work. Western blotting was performed using antibodies against REST (Abcam, Cambridge, UK), doublecortin (DCX; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or β-actin (Santa Cruz Biotechnology). The blots were developed using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA) and digitally scanned (GS-700; Bio-Rad, Hercules, CA, USA). The optical density of each band relative to that of the β-actin band was determined using Molecular Analyst® software (Bio-Rad). For real-time PCR, total RNA was isolated from each hemisphere using QIAshredder and RNeasy kits (Qiagen, Valencia, CA, USA). miR-124 levels were measured using the mirVana qRT-PCR miRNA Detection Kit and TaqMan miRNA assays (Ambion, Applied Biosystems).
Doublecortin dcx
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom, United States
Doublecortin (DCX) is a protein that plays a crucial role in the migration and differentiation of neurons during brain development. It is primarily expressed in neuronal precursor cells and is a widely used marker for identifying and studying these cells.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (2 mentions)
Neuronal nuclei (neun), by Merck Group (2 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (2 mentions)
Rneasy kit, by Qiagen (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Mirvana qrt pcr mirna detection kit, by Thermo Fisher Scientific (1 mentions)
Gs 700, by Bio-Rad (1 mentions)
Molecular analyst software, by Bio-Rad (1 mentions)
β actin, by Bio-Rad (1 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Synapsin 1, by Abcam (1 mentions)
Bromodeoxyuridine (brdu), by Santa Cruz Biotechnology (1 mentions)
Rabbit anti dcx antibody, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Nestin, by Abcam (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Hybond ecl, by Cytiva (1 mentions)
13 protocols using doublecortin dcx
1
Exosome Injection Alters Brain Molecular Profiles
2
Adult Mouse Neural Progenitor Cell Isolation and Assays
3
Immunohistochemical Analysis of Neurogenesis
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Mice were transcardially perfused with 4% paraformaldehyde in
phosphate-buffered saline and brains were carefully dissected. Postfixation
was carried out in 4% buffered formalin at 4 °C before
paraffin embedding. Immunohistochemistry on 4 μm sagittal paraffin
and frozen sections was performed as described previously.29 (link), 31 (link)The following antibodies were used: 4G8 (Aβ17-14, 1:1,0000, Covance,
Princeton, NJ, USA); and doublecortin (DCX; 1:200, Santa Cruz, Dallas, TX,
USA).
phosphate-buffered saline and brains were carefully dissected. Postfixation
was carried out in 4% buffered formalin at 4 °C before
paraffin embedding. Immunohistochemistry on 4 μm sagittal paraffin
and frozen sections was performed as described previously.29 (link), 31 (link)The following antibodies were used: 4G8 (Aβ17-14, 1:1,0000, Covance,
Princeton, NJ, USA); and doublecortin (DCX; 1:200, Santa Cruz, Dallas, TX,
USA).
4
Immunofluorescent Staining of Brain Tissue
5
Quantifying Hippocampal Neurogenesis in Rats
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Six-month-old male F344 rats acquired from Harlan Sprague-Dawley were employed for this experiment. Each animal received IN administration of either 100 × 109 EVs (EVs group, n = 6) or SEC buffer (VEH group, n = 6), as described in the previous section. Thirteen days later, the animals received three injections of 5ʹ-bromodeoxyuridine (BrdU, once every 8 hours at 75 mg/Kg). Eight hours later, the animals were euthanized through intracardiac perfusions with 4% paraformaldehyde, and the brain tissues were processed for cryostat sectioning as described in our previous reports [36 (link),37 (link)]. Thirty-micrometre-thick serial sections (every 15th) through the entire septotemporal axis of the hippocampus were immunohistochemically processed for identifying BrdU (1:100; Sigma), doublecortin (DCX, 1:500; Santa Cruz) and Ki-67 (1:500; Millipore) [1 (link),38 (link)]. The number of newly born cells (BrdU+ cells), newly born neurons (DCX+ cells) and proliferating cells (Ki-67+ cells) in the entire subgranular zone-granule cell layer (SGZ-GCL) were quantified using stereology [39 (link),40 (link)]. Additional serial sections were also processed for BrdU-DCX dual immunofluorescence and Z-section analysis in a confocal microscope to measure the extent of neuronal differentiation of newly born cells and to calculate net hippocampal neurogenesis [38 (link)].
6
Quantifying Hippocampal Neurogenesis via DCX
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After transcardiac perfusion with phosphate buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde (PFA), brains were removed, postfixed for 4 h in PFA, cryoprotected in 30% sucrose for 24 h, snap frozen in liquid nitrogen, and stored at -80°C before sectioning. Free-floating (30 μm) coronal sections containing the hippocampus (from -0.9 mm to -3.1 mm relative to bregma) were collected on a cryostat for immunohistochemistry.
Neurogenesis in the hippocampus was evaluated by determining the number of immature neurons in the dentate gyrus characterized by the endogenous marker doublecortin (DCX) (Santa Cruz Biotechnology, CA, USA), a cytoplasmic protein expressed transiently in newborn neurons only [38 (link),39 (link)]. Briefly, 30-μm sections were incubated with rabbit anti-DCX antibody (1:800; Abcam, Cambridge, UK) followed by a biotinylated secondary antibody and streptavidin peroxidase complex, which was visualized by diaminobenzidine-nickel staining. The number of DCX positive cells was counted on three sections representing the same three levels of the dentate gyrus for all animals [39 (link)].
Neurogenesis in the hippocampus was evaluated by determining the number of immature neurons in the dentate gyrus characterized by the endogenous marker doublecortin (DCX) (Santa Cruz Biotechnology, CA, USA), a cytoplasmic protein expressed transiently in newborn neurons only [38 (link),39 (link)]. Briefly, 30-μm sections were incubated with rabbit anti-DCX antibody (1:800; Abcam, Cambridge, UK) followed by a biotinylated secondary antibody and streptavidin peroxidase complex, which was visualized by diaminobenzidine-nickel staining. The number of DCX positive cells was counted on three sections representing the same three levels of the dentate gyrus for all animals [39 (link)].
7
Protein Expression Analysis in Neuronal Cells
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β-actin (Sigma Life Science, Cat#A2228, 1:1000, RRID: AB_476697), Tau (Abcam, Cat# ab75714, 1:500, RRID:AB_1310734), phosphorylated TRPV1 (Thermo Fisher Scientific, Cat# PA5–64860, 1:500, RRID:AB_2663797), Sigma-1R (ProteinTech, Cat# 15168–1-AP, 1:1,000, RRID:AB_2301712), Doublecortin (DCX) (Santa cruz, Cat# sc-271390, 1:1000, RRID:AB_10610966).
8
Western Blot Analysis of Neural Markers
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Hippocampal tissues were homogenized and centrifuged at 13,000 g at 4 °C for 10 min. Proteins in the supernatant were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences, Little Chalfont, UK). Membranes were blocked and incubated overnight at 4 °C with primary antibodies against octamer-binding transcription factor 4 (OCT4; 1:200), doublecortin (DCX; 1:1000), or β-actin (1:200) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); against SOX2 (1:200), Ki67 (1:1000), nestin (1:1000), or brain lipid-binding protein (BLBP; 1:800) (all from Abcam, Cambridge, MA, USA); or against glial fibrillary acidic protein (GFAP; 1:1000; EMD Millipore, Billerica, MA, USA). After thorough washing, blots were incubated with secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) conjugated with horseradish peroxidase. Protein bands were detected using enhanced chemiluminescence (Amersham Biosciences) and quantitated using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Band intensities were normalized to β-actin.
9
Multifaceted Analysis of Brain Lesions
10
Immunofluorescent Labeling of Neural Markers
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Cells were passaged into 24-well plates containing poly D-lysine (50 μg/ml) coated glass coverslips and after experimental observation were fixed with 4 % paraformaldehyde (PFA), rinsed and incubated overnight in primary antibody. After further rinsing with PBS, cells were incubated in the appropriate secondary fluorescent antibody at 1:200 dilution in PBS with 0.3 % Triton X (1 h) followed by rinsing and mounting the coverslips with Vectashield® fluorescent mounting medium.
Primary antibodies used were: DCC 1:500 (BD Pharmingen, Oxford, UK, #554223); doublecortin (DCX) 1:200 (Santa Cruz, California; #SC8066), neogenin 1:500 (Santa Cruz, California; #SC6536).
Secondary antibodies (all 1:200) were AlexaFluor 594 goat anti-mouse #A11032; AlexaFluor 488 goat anti-rabbit #A11008; AlexaFluor rabbit anti-goat #A11078 (Life Technologies, Paisley, UK).
Primary antibodies used were: DCC 1:500 (BD Pharmingen, Oxford, UK, #554223); doublecortin (DCX) 1:200 (Santa Cruz, California; #SC8066), neogenin 1:500 (Santa Cruz, California; #SC6536).
Secondary antibodies (all 1:200) were AlexaFluor 594 goat anti-mouse #A11032; AlexaFluor 488 goat anti-rabbit #A11008; AlexaFluor rabbit anti-goat #A11078 (Life Technologies, Paisley, UK).
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