Immunocytochemistry and immunostaining were conducted according to published approaches (Guo et al., 2011 (link), Liu et al., 2010 (link), Liu et al., 2013a (link)). For immunostaining cultured cells, anti-neuron-specific type β-III tubulin (Biolegend, #801202; 1:1,000), anti-glial fibrillary acidic protein (Millipore, MAB377; 1:1,000), anti-BrdU (Abcam, ab6326; 1:2000), and anti-cleaved caspase 3 (Cell Signaling, #9664; 1:200) were used as the primary antibodies. For immunohistochemistry staining, the primary antibodies used were as follows: anti-Ki67 (Thermo Fisher, RM9106; 1:1,000), anti-BrdU (Abcam, ab6326; 1:1,000), and anti-DCX (Cell Signaling, #4604; 1:500). The secondary antibodies conjugated to Alexa Fluor 488 or 594 with a concentration of 1:500 were used at room temperature. To analyze the amount of BrdU+GFP+, or BrdU+DCX+GFP+ cells in the DG, we used one in six series of 40-μm brain sections starting at beginning of hippocampus (relative to bregma, −1.5 mm) to the end of hippocampus (relative to bregma, −3.5 mm). For details, see Supplemental Experimental Procedures .
Anti dcx
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-DCX is a primary antibody product manufactured by Cell Signaling Technology. It is designed to detect doublecortin (DCX), a microtubule-associated protein that plays a crucial role in neuronal migration and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Anti brdu, by Abcam (3 mentions)
Anti neun, by Merck Group (2 mentions)
Ab6326, by Cell Signaling Technology (1 mentions)
Ab6326, by Abcam (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Mab377, by Abcam (1 mentions)
Anti sox2, by Abcam (1 mentions)
Dl 1174, by Vector Laboratories (1 mentions)
Anti tuj1, by R&D Systems (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Anti neun, by Abcam (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Anti tubb3, by BioLegend (1 mentions)
Axio scan z1 slide scanner, by Zeiss (1 mentions)
Anti ki67, by Cell Signaling Technology (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti dcx
1
Immunocytochemistry and Immunostaining Protocol
2
Immunofluorescence Staining of Brain Tissue
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Brain tissue slices (10-μm) or cultured cell samples were treated with 4% PFA for 20 min and stored at room temperature. After three washes with PBS, all slides were incubated in blocking buffer (3% bovine serum albumin, 5% normal donkey serum, and 0.3% Triton X-100 in PBS) for 1 h at room temperature. Slides or coverslips were immersed in primary antibody buffer (200 μL per slide or 40 μL per coverslip) for incubation overnight at 4°C. Primary antibodies used in this study include: anti-GFAP (1:1000; Dako, Glostrup, Denmark), anti-Tuj1 (1:500; R&D Systems, Minneapolis, MN, USA), anti-DCX (1:400; Cell Signaling Technology, Danvers, MA, USA), anti-BrdU (1:200; Abcam, Cambridge, UK), anti-Sox2 (1:200, Abcam), and anti-NeuN (1:300; Abcam). The following day, coverslips and slides were washed three times with PBS, and then incubated with appropriate Alexa488-, Alexa568- or Cy3-conjugated secondary antibodies (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature, or lectin-FITC (Dylight, DL-1174, Vectorlabs, CA, USA) for 2 h. DAPI staining for 15 min was used to label nuclei.
3
Neurogenesis Analysis in Mouse Brains
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Animals were sacrificed by intracardial perfusion with saline, and mice brains were fixed with 4% paraformaldehyde. The fixed brains were dehydrated once in 20% sucrose and twice in 30 percent sucrose (both in PBS). The coronal sections were cut to a thickness of 35 μm, and the sections were washed with 1x PBS. The sections were blocked with blocking buffer (1% BSA+0.3% Triton X-100+10% goat serum in PBS) for one hour at room temperature after three washes in 1x PBS. The coronal sections were then incubated with the primary antibody anti-DCX: (1 : 400, Cell Signaling Technology, #14082) for an additional overnight period at 4°C, followed by an additional two hours at room temperature with the fluoroconjugated secondary antibodies. Sections were washed three times and stained with DAPI (4′,6-diamidino-2-phenylindole) solution. Finally, images were captured with a Leica TCS SP8 confocal microscope (Leica Microsystems, Germany).
4
Multicolor Immunofluorescence Staining Protocol
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Cell samples were fixed at a final concentration of 4% low methanol formaldehyde solution (product number 4235.4, Roth). Samples were then incubated in blocking buffer (DPBS with 10% goat serum, product number G923, Merck, and 0.1% Triton X-100, product number HFH10, Invitrogen) for 1 h. Primary antibodies were incubated in dilution buffer (DPBS with 1% goat serum and 0.01% Triton X-100) overnight at 4 °C. Secondary antibodies were incubated in the dilution buffer for 1 h at room temperature. Nuclear staining was performed with Hoechst (1:2000 in dilution buffer) for 10 min. After each incubation step, the samples were washed three times with DPBS. The following antibodies were used: anti-TUBB3 (1:500, product number 801201, Biolegend), anti-TH (1:500, product number P40101, PelFreez), anti SYN1 (1:500, product number AB1543, Merck), anti DCX (1:800, product number 4604S, Cell Signalling), anti-rabbit Alexa Fluor 488 (1:1000, product number A21206, Invitrogen), anti-rabbit CF594 (1:1000, product number SAB4600107, Merck) and CF633 anti-mouse (1:1000, product number A-21136, Invitrogen). A Nikon CrEST X-light V3 spinning disc was used to image the samples.
5
Immunohistochemistry Analysis of Aged Brains
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This procedure has been previously described (Criado-Marrero et al., 2019 (link)). Briefly, after behavioral training, mice were euthanized with a Somnasol overdose at 150 (young) or 300 (aged) days of age. Following transcardial perfusion with 0.9% saline, brains were collected and fixed in 4% paraformaldehyde. Following cryopreservation, tissue was sectioned into horizontal slices at 25 and 50 μm for IHC and stereological analyses, respectively. Free-floating sections were stained as previously described (Blair et al., 2013 (link); Dickey et al., 2009 (link)) using the following primary antibodies: anti-Ki-67 (Cell Signaling, Danvers, Massachusetts, USA, CS-9449; 1:1000), anti-DCX (Cell Signaling, Danvers, Massachusetts, USA,CS-4604; 1:300), anti-GFAP (Millipore, Burlington, MA, USA, MAB360, 1:10,000), anti-Iba1 (WAKO, Richmond, VA, USA, 016-26461, 1:10,000). Images were taken at 20× using a Zeiss AxioScan.Z1 slide scanner (Zeiss, Oberkochen, Germany) and tissue segmentation was performed using the NearCYTE software (www.nearcyte.org ).
6
Immunohistochemistry of Neurogenesis Markers
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After three washes with TBS, free-floating sections were incubated for 30 min with HCl 2N at 37°C, followed by two washes with boric acid (0.1M; pH=8.9; 10 min/wash) for antigen retrieval. The sections were incubated with the blocking solution (TBS + Triton X 0,025% + BSA 0.1%) for 4 hours, and then incubated with anti-BrdU (Abcam; Cambridge, England, UK; rabbit; 1:100), anti-DCX (Cell-Signaling; rat; 1:500) and anti-NeuN (Millipore; MA, USA; mouse; 1:500) primary antibodies overnight at 18°C under agitation. On the next day, the sections were washed three times with TBS-T (Tris-buffered saline with 10% Triton) and incubated for 1.5h with the secondary antibodies (anti-rabbit AlexaFluor 488, anti-rat Alexa-Fluor 594, and anti-mouse AlexaFluor 647; Invitrogen, Massachusetts, USA).
7
Characterizing Neuronal Protein Expression
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Standard immunocytochemistry and Western blot techniques were used to characterize cell type–specific markers and phenotypically relevant protein levels. The following antibodies were used for immunostaining according to the manufacturers’ instructions: anti-GFP (#1010; Aves Labs Inc), anti-PAX6 (PRB-278P; Covance), anti-Nestin (PRB-315C; Covance), anti-Map2 (#M2320; Sigma-Aldrich), and anti-Ctip2 (ab18465; Abcam). The following antibodies were used for Western blot assays: anti-Shank3 (sc30193; Santa Cruz), anti-Shank3 (sc 23547; Santa Cruz), anti-PSD95 (ab2723; Abcam), anti-Homer (#160011; Synaptic Systems), anti-NLGN1 (SAB1407153; Sigma-Aldrich), anti-PIKE (#07-675; Upstate), anti-Akt (#4691S; Cell Signaling), anti-phospho Akt (#4058; Cell Signaling), anti-mTOR (#4691; Cell Signaling), anti-phospho mTOR (#2971; Cell Signaling), anti-Jip2 (N135/37; NeuroMab), anti-NRP1 (SAB1411572; Sigma-Aldrich), anti-JNK1/3 (sc474; Santa Cruz), anti-phosphor JNK (#V793A; Promega), anti-DCX (#4604; Cell Signaling), anti-NeuN (#MAB377; EMD Millipore), anti-Tuj1 (#903401; BioLegend), anti-GAPDH (sc 47724; Santa Cruz), and anti-Actin (sc1616; Santa Cruz). Western blot quantification was performed using ImageJ.
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