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Doublecortin

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Doublecortin is a microtubule-associated protein that plays a crucial role in the migration and differentiation of neuronal precursor cells during brain development. It is a widely used marker for identifying and labeling immature neurons.

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Lab products found in correlation

Bromodeoxyuridine (brdu), by Abcam (5 mentions) Neuronal nuclei (neun), by Cell Signaling Technology (5 mentions) Alexa 488, by Thermo Fisher Scientific (5 mentions) Alexa 555, by Thermo Fisher Scientific (5 mentions) Il 1β, by R&D Systems (5 mentions) Alexa 647, by Thermo Fisher Scientific (4 mentions) Synaptophysin, by Synaptic Systems (4 mentions) Glial fibrillary acidic protein (gfap), by Thermo Fisher Scientific (3 mentions) D3s3i, by Abcam (2 mentions) Mash1, by BD (2 mentions) Glial fibrillary acidic protein (gfap), by BD (2 mentions) C fos, by Merck Group (2 mentions) Phospho stat3, by Abcam (2 mentions) Nlrp3, by R&D Systems (2 mentions) Caspase 1, by Cell Signaling Technology (2 mentions) Lsm 510 meta confocal microscope, by Zeiss (2 mentions) Ab97959, by Cell Signaling Technology (1 mentions) Ab18258, by Merck Group (1 mentions) Mab1637, by Merck Group (1 mentions) Mab5326, by Abcam (1 mentions)

10 protocols using doublecortin

1

Immunolabeling of NPCs and Neurons

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Immunolabeling of NPCs and derived neurons was accomplished by utilization of a standard two-day protocol with: 15 min fixation in 4% PFA, 10 min permeabilization in 0.1% triton-PBS and 30 min blocking with 5% donkey serum. Following dilutions of antihuman primary antibodies in PBS were applied overnight at 4 °C: Nestin (Millipore, MAB5326, 1:200), SOX2 (Abcam, ab97959, 1:200), Doublecortin (Cell Signaling, 4604S, 1:200), MAP2 (Millipore, MAB3418, 1:200), PSA-NCAM (Millipore, MAB5324, 1:200), PSD95 (Abcam, ab18258, 1:200) Tubulin, beta 3 (Millipore, MAB1637, 1:200). For fluorescence staining, we incubated samples for 2 h with Alexa-488 and Alexa-594 conjugated donkey antibodies (1:200) against mouse and rabbit immunoglobulins (Supplementary Figs 6 and 7 are representative images from two sets of immunolabelled cultures of NPCs and neurons).
Jiang X., Chen J., Bajić A., Zhang C., Song X., Carroll S.L., Cai Z.L., Tang M., Xue M., Cheng N., Schaaf C.P., Li F., MacKenzie K.R., Ferreon A.C., Xia F., Wang M.C., Maletić-Savatić M, & Wang J. (2017). Quantitative real-time imaging of glutathione. Nature Communications, 8, 16087.
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2

Immunohistochemical Analysis of Alzheimer's Pathology

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The cryoprotected hemibrain tissue was serially cut at 50 μm on a freezing microtome. Serial sections of 500 μm apart spanning the whole hemibrain were immunostained with antibodies to Aβ1–40 and Aβ1–42 (Invitrogen, Grand Island, NY #44348A, #44-344) to define Aβ deposits and to doublecortin (Cell Signaling Technology Inc., MA), as a marker of neurogenesis. Immunohistochemical procedures were performed as previously described (Kowall et al., 2000 (link)). In brief, free-floating sections were incubated overnight in primary antibody followed by PBS (Phosphate buffered saline) washes and incubation in peroxidase-conjugated secondary antibody followed by development using 3,3′-diaminobenzidine tetrahydrochloride (DAB) as a chromogen.
Aytan N., Choi J.K., Carreras I., Crabtree L., Nguyen B., Lehar M., Blusztajn J.K., Jenkins B.G, & Dedeoglu A. (2018). Protective effects of 7,8-dihydroxyflavone on neuropathological and neurochemical changes in a mouse model of Alzheimer’s disease. European journal of pharmacology, 828, 9-17.
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3

Immunohistochemical Profiling of ZIKV and WNV

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The following primary antibodies were used for IHC analyses: WNV (1:100, polyclonal25 (link)), ZIKV (1:50; gift from M. Diamond (St. Louis, MO), ZV-13), NeuN (1:1,000; Cell Signaling, cat no. 12943S, clone D3S3I), BrdU (1:200; Abcam, cat. no. ab1893, polyclonal), doublecortin (1:125; Cell Signaling, cat. no. 4604S, polyclonal), GFAP (1:250; Thermo, cat. no. 13–0300, clone 2.2B10), IL-1β (1:100; R&D, cat. no. AF-401, polyclonal), CD3 (1:250; R&D, cat. no. MAB4841, clone 17A2), CD11c (1:50; Novus, cat. no. NB110– 97871, clone AP-MAB0806), GFP (1:500; Abcam, cat. no. ab13970, polyclonal), IBA1 (1:200; Wako, cat. no. 019–19741, polyclonal), TMEM119 (1:200; Abcam, cat. no. ab209064, clone 28–3), TUNEL (Roche In situ Cell Death kit Red), and synaptophysin (1:250; Synaptic Systems, cat. no. 101004, polyclonal). Secondary antibodies conjugated to Alexa-488 (Invitrogen, cat. no. A-21206, polyclonal) or Alexa-555 (Invitrogen, cat. no. A-21435, polyclonal) were used at a 1:400 dilution.
Garber C., Soung A., Vollmer L.L., Kanmogne M., Last A., Brown J, & Klein R.S. (2019). T cells promote microglia-mediated synaptic elimination and cognitive dysfunction during recovery from neuropathogenic flaviviruses. Nature neuroscience, 22(8), 1276-1288.
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4

Multiplex Immunofluorescence Staining Protocol

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WNV (1:100 described previously8 (link)), NeuN (1:100, Cell Signaling, Cat 12943S, Clone D3S3I), BrdU (1:200, Abcam, Cat ab1893, polyclonal), CD45 (Biolegend, Cat 103114, Clone 30-F11), Doublecortin (1:150, Cell Signaling, Cat 4604S, polyclonal), GFAP (1:50 for flow cytometry; 1: 200 for IHC, BD, Cat 561483, Clone 1B4), IL-1β (R&D, Cat AF-401, polyclonal), Mash1 (BD, Cat 556604, Clone 24B72D11.1), Ki67 (Abcam, Cat AB15580, polyclonal), Synaptophysin (1:250, Synaptic Systems, Cat 101004, polyclonal). Secondary antibodies conjugated to Alexa-488, Alexa-555, or Alexa-647 (Invitrogen) were used at a1:400 dilution.
Garber C., Vasek M.J., Vollmer L.L., Sun T., Jiang X, & Klein R.S. (2018). Astrocytes decrease adult neurogenesis during virus-induced memory dysfunction via interleukin-1. Nature immunology, 19(2), 151-161.
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5

Antibody Panel for Neural Cell Studies

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The PIWIL1 antibody for Western blotting (1:1,000) has been described previously [29 (link)]. Other antibodies for immunohistochemistry or Western blotting were Tbr2 (rabbit, 1:500; Abcam, Cambridge, UK), green fluorescent protein (GFP) (chicken, 1:1,000; Abcam, Cambridge, UK), BrdU (mouse, 1:200; Sigma, St. Louis, MO), Sox2 (goat, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA), MAP2 (rabbit, 1:1,000; Chemicon, Bioscience Research Reagents, Temecula, CA), Nestin (mouse, 1:100; Millipore, Billerica, MA), doublecortin (rabbit, 1:1,000; Cell Signaling Technology [CST], Danvers, MA), Flag (rabbit, 1: 1,000; Cell Signaling Technology [CST], Danvers, MA), Cux1 (Rabbit, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), and Tbr1 (Rabbit, 1:500; Abcam, Cambridge, UK).
Zhao P.P., Yao M.J., Chang S.Y., Gou L.T., Liu M.F., Qiu Z.L, & Yuan X.B. (2015). Novel function of PIWIL1 in neuronal polarization and migration via regulation of microtubule-associated proteins. Molecular Brain, 8, 39.
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6

Multiplex Immunofluorescence Staining Protocol

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WNV (1:100 described previously8 (link)), NeuN (1:100, Cell Signaling, Cat 12943S, Clone D3S3I), BrdU (1:200, Abcam, Cat ab1893, polyclonal), CD45 (Biolegend, Cat 103114, Clone 30-F11), Doublecortin (1:150, Cell Signaling, Cat 4604S, polyclonal), GFAP (1:50 for flow cytometry; 1: 200 for IHC, BD, Cat 561483, Clone 1B4), IL-1β (R&D, Cat AF-401, polyclonal), Mash1 (BD, Cat 556604, Clone 24B72D11.1), Ki67 (Abcam, Cat AB15580, polyclonal), Synaptophysin (1:250, Synaptic Systems, Cat 101004, polyclonal). Secondary antibodies conjugated to Alexa-488, Alexa-555, or Alexa-647 (Invitrogen) were used at a1:400 dilution.
Garber C., Vasek M.J., Vollmer L.L., Sun T., Jiang X, & Klein R.S. (2018). Astrocytes decrease adult neurogenesis during virus-induced memory dysfunction via interleukin-1. Nature immunology, 19(2), 151-161.
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7

Doublecortin Expression After Traumatic Brain Injury

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We evaluated protein expression levels 21 days after TBI in the hippocampal dentate gyrus of mice. Slides of mouse brains were washed with PBS, immersed in 0.5% Triton-X-100 for 20 min, and then blocked with 1% normal goat serum in PBS containing 0.1 % Tween 20 for 40 min at room temperature. Sections were then diluted with the primary rabbit polyclonal antibody Doublecortin (1:200, #4604, Cell Signaling Technology) and incubated overnight at 4° C. After rinsing with PBS, the specimen sections were incubated with Anti-Rabbit IgG (H+L) (1:500 A11034, Alexa Fluor 488 Conjugate) for 1 h at room temperature in the dark. Slides were washed in PBS, mounted with mounting medium with DAPI (H-1800, Vector Laboratories), and sealed with coverslips for analysis. Images were captured using a fluorescence microscope. In vitro, cells were discarded from the medium, washed with PBS, and fixed with 4% paraformaldehyde at room temperature. Cells were permeabilized using 0.1% Triton-X100, followed by blocking with 2% bovine serum albumin. Cells were incubated with diluted primary antibody mouse monoclonal antibody MAP-2 (1:500, sc-74421, Santa Cruz) overnight at 4° C. After washing with PBST, diluted secondary antibodies were added for 1 h and then mounted with DAPI for fluorescence microscopy.
Chen Y.H., Chen Y.C., Chin Y.T., Wang C.C., Hwang L.L., Yang L.Y, & Lu D.Y. (2022). 2, 3, 5, 4’-tetrahydroxystilbene-2-O-beta-D-glucoside protects against neuronal cell death and traumatic brain injury-induced pathophysiology. Aging (Albany NY), 14(6), 2607-2627.
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8

Culturing and Characterizing Neural Progenitor Cells

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Neural progenitor cells derived from control dams were expanded as adherent monolayer cultures under either proliferating or differentiating conditions and were prepared for immunohistochemical analysis and imaging. Cells were transferred to poly-D-lysine coated coverslips after the second passage and grown in complete medium with growth factors EGF and FGF-2. After 2 days, cells were immunostained with the neural stem cell marker Nestin (1:1000; BD Biosciences) and counterstained with DAPI (0.5 μg/mL; Sigma) (Roitbak et al., 2011 (link)). To determine appropriate culture of neurons, cells were transferred to poly-D-lysine coated plates after the second passage and grown in complete medium without growth factors. After differentiation for 7 days without growth factors, cells were immunostained for astrocytes with GFAP (1:1000; Sigma-Aldrich) and neuroblasts and immature neurons were immunostained with doublecortin (1:1500; Cell Signaling) and counterstained with DAPI (0.5 μg/mL; Sigma). Secondary antibodies used for analysis included Alexa Fluor 647 (1:250; Invitrogen; A-21236) and Cy3 (1:500; Invitrogen; A10520). Images for publication were acquired using a Zeiss LSM-510 META confocal microscope equipped with a laser diode, one argon laser, and two HeNe lasers. Maximum intensity projections of Z stacks were acquired with a 20X objective.
Tyler C.R, & Allan A.M. (2014). Prenatal alcohol exposure alters expression of neurogenesis-related genes in an ex vivo cell culture model. Alcohol (Fayetteville, N.Y.), 48(5), 483-492.
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9

Comprehensive Immunohistochemical Analysis of Neurological Markers

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The following primary antibodies were used for IHC analyses: NeuN (1:1000; Cell Signaling, cat no. 12943S, clone D3S3I), BrdU (1:250; Abcam, cat. no. ab1893, polyclonal), doublecortin (1:125; Cell Signaling, cat. no. 4604S, polyclonal), GFAP (1:250; Thermo, cat. no. 13–0300, clone 2.2B10), IL-1β (1:100; R&D, cat. no. AF-401, polyclonal), IBA1 (1:500; Synaptic Systems, cat. no. 234006, polyclonal), Homer (1:200; Synaptic Systems, cat. no. 160002, polycloncal), Sox9 (1:500; Millipore, cat no. AB5535, polyclonal), c-Fos (1:1000; Millipore, cat. no. ABE457, polyclonal), Caspase-1 (1:200, Cell Signaling, cat. no. 24232S, monoclonal), NLRP3 (1:200, R&D Systems, cat. no. MAB7578, monoclonal), synaptophysin (1:250; Synaptic Systems, cat. no. 101004, polyclonal), and phospho-STAT3 (1:100, Abcam, cat. no. ab76315, monoclonal). Secondary antibodies conjugated to Alexa-488, Alexa-555, or Alexa-647 (Invitrogen, polyclonal) were used at a 1:500 dilution.
Soung A.L., Davé V.A., Garber C., Tycksen E.D., Vollmer L.L, & Klein R.S. (2021). IL-1 reprogramming of adult neural stem cells limits neurocognitive recovery after viral encephalitis by maintaining a proinflammatory state. Brain, behavior, and immunity, 99, 383-396.
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10

Comprehensive Immunohistochemical Analysis of Neurological Markers

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The following primary antibodies were used for IHC analyses: NeuN (1:1000; Cell Signaling, cat no. 12943S, clone D3S3I), BrdU (1:250; Abcam, cat. no. ab1893, polyclonal), doublecortin (1:125; Cell Signaling, cat. no. 4604S, polyclonal), GFAP (1:250; Thermo, cat. no. 13–0300, clone 2.2B10), IL-1β (1:100; R&D, cat. no. AF-401, polyclonal), IBA1 (1:500; Synaptic Systems, cat. no. 234006, polyclonal), Homer (1:200; Synaptic Systems, cat. no. 160002, polycloncal), Sox9 (1:500; Millipore, cat no. AB5535, polyclonal), c-Fos (1:1000; Millipore, cat. no. ABE457, polyclonal), Caspase-1 (1:200, Cell Signaling, cat. no. 24232S, monoclonal), NLRP3 (1:200, R&D Systems, cat. no. MAB7578, monoclonal), synaptophysin (1:250; Synaptic Systems, cat. no. 101004, polyclonal), and phospho-STAT3 (1:100, Abcam, cat. no. ab76315, monoclonal). Secondary antibodies conjugated to Alexa-488, Alexa-555, or Alexa-647 (Invitrogen, polyclonal) were used at a 1:500 dilution.
Soung A.L., Davé V.A., Garber C., Tycksen E.D., Vollmer L.L, & Klein R.S. (2021). IL-1 reprogramming of adult neural stem cells limits neurocognitive recovery after viral encephalitis by maintaining a proinflammatory state. Brain, behavior, and immunity, 99, 383-396.
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