A K643A variant of human DISC1 or a K640A variant of mouse DISC1 (mDISC1) was introduced by PCR-based mutagenesis as mentioned previously [25 (link)]. All constructs in the present study were created as described previously [24 (link), 32 (link)]. We used short hairpin RNA (shRNA) construct of DISC1 that was published previously (targeting sequence is 5'-GGCAAACACTGTGAAGTGC-3') [24 (link)]. We used a control RNAi construct with a scrambled sequence without homology to any known messenger RNA [33 (link)]. GFP-tagged SUMO1, FLAG-tagged SUMO1, and Myc-tagged Ubc9 constructs were generous gifts from Dr. Pandolfi (Beth Israel Deaconess Cancer Center, USA), Dr. Hayward (Johns Hopkins University, USA), and Dr. Matunis (Johns Hopkins School of Public Health, USA), respectively. The following antibodies were used: mouse monoclonal anti-HA antibody (Covance); rabbit polyclonal anti-FLAG antibody (Sigma); rabbit polyclonal anti-SUMO1 antibody (Zymed); mouse monoclonal anti-BrdU antibody (BD Biosciences); anti-DISC1-C2 antibody [34 (link)]; and a rabbit polyclonal anti-human DISC1 antibody (kindly provided by Dr. Akiyama, University of Tokyo) [35 (link)–37 (link)].
Mouse anti brdu monoclonal antibody
Manufactured by BD
Sourced in United States
The mouse anti-BrdU monoclonal antibody is a laboratory reagent used to detect the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into cellular DNA. This antibody specifically binds to BrdU, allowing the labeling and identification of proliferating cells during DNA synthesis.
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Lab products found in correlation
Rat anti brdu monoclonal antibody, by Bio-Rad (7 mentions)
Alexa fluor 594 goat anti rat antibodies, by Thermo Fisher Scientific (4 mentions)
Alexafluor 555 conjugated goat anti rat igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 rabbit anti mouse, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Goat serum, by Abcam (2 mentions)
Vector novared peroxidase hrp substrate kit, by Vector Laboratories (2 mentions)
Harris haematoxylin solution, by Merck Group (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (2 mentions)
Bromodeoxyuridine (brdu), by Merck Group (2 mentions)
Rat monoclonal anti brdu antibody, by Abcam (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Radiance confocal microscope, by Bio-Rad (2 mentions)
Kaluza software, by BD (2 mentions)
Fitc conjugated anti mouse antibody, by Merck Group (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
7 aad staining, by BD (2 mentions)
29 protocols using mouse anti brdu monoclonal antibody
1
Genetic manipulation of DISC1 in cells
2
Immunohistochemical Analysis of BrdU Incorporation
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After the fluorescence in situ hybridization, sections were transferred to PBS, incubated in 2 M HCl for 30 min at room temperature and washed three times in PBS. After blocking reaction in PBS containing 1.0% BSA and 0.25% Tween 20, sections were incubated with mouse monoclonal anti-BrdU antibody (BD Bioscience San Jose, CA, USA) diluted to 2.5 μg/ml in PBS containing 1.0% BSA and 0.25% Tween 20 overnight at 4 °C. Control sections were incubated with mouse normal IgG (Chemicon Europe) instead of anti-BrdU antibody. Sections were then washed four times in PBS, incubated with Alexa 555-conjugated goat anti-mouse antibody (Invitrogen) diluted to 1:1000 in PBS containing 1.0% BSA and 0.25% Tween 20 for 2 h at room temperature and washed four times in PBS. After counterstaining with 30 mM DAPI in water, the signals were examined under the fluorescence microscope (Carl Zeiss). The images were merged using Adobe Photoshop CS4.
3
Biochemical and Electrophysiological Analysis of TREK-1 and BDNF Signaling
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The ELISA kit to measure corticosterone was from Enzo Life Science. Rabbit polyclonal antibodies anti-TREK-1 and anti-proBDNF were from Alomone Labs (Israel). Rabbit polyclonal antibodies anti-furin and anti-NaKATPase, the mouse monoclonal anti-beta-actin and goat anti-TGN38 and anti-DCX antibodies were from SantaCruz Technologies (United States). Rabbit polyclonal antibodies anti-TrkB and anti-phospho-(Tyr 705)-TrkB were from Signalway Antibody (United States). Rabbit polyclonal antibodies against BDNF and plasminogen were from GeneTex (United States). Mouse monoclonal anti-BrdU antibody was from Becton Dickinson (United States). Anti-rabbit, anti-mouse, and anti-goat secondary antibodies were from Cell Signaling Technologies. HRP substrate (Immobilon Forte, Millipore), Nitrocellulose (BioTrace NT, Life Sciences), SDS-PAGE gels and Mini Protean apparatus were from Biorad. BrdU was from Interchim (France). Neurobasal medium, B-27 and Penicillin-Streptomycin were from Thermo Fisher Scientific. Poly-D-Lysine was from Sigma-Aldrich. Patch clamp was performed on a pClamp 8.2 apparatus (Molecular Devices)
4
Quantifying BrdU-positive Neurons in Mouse Hippocampus
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One day after 5-bromo-2′-deoxyuridine (BrdU) injections, 12 mg per animal divided in four bolus of 300 μL injected every 2 h, mice were anesthetized with isoflurane and transcardially perfused with 20 mL of NaCl 0.9 % followed by 20 mL paraformaldehyde 4 %/NaCl 0.9 %. By using a vibratome (Leica), brains were cut into 40-μm sections, throughout the entire hippocampus. Eight slices, from bregma 3.3 to bregma 5.3, were retained to process the BrdU immunohistochemistry as previously described (Heurteaux et al. 2006b (link)). For each BrdU labeling, slices were first incubated with a mouse monoclonal anti-BrdU antibody (1/8,000, Becton Dickinson). For chromogenic immunodetection, sections were incubated during 2 h in biotin-conjugated species-specific secondary antibodies (1/400; Vector laboratories) followed by a peroxidase-avidin complex solution, to amplify the reaction. The peroxidase activity of immune complex was visualized with DAB staining using the VectaStain ABC kit according to the manufacturer’s protocol (Vector Laboratories).
5
Immunohistochemical Assessment of Cell Proliferation
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Liver, pancreas, kidney and heart sections were fixed in 10% of buffered formalin and processed for staining with haematoxylin‐eosin (H&E) or immunohistochemistry (IHC). For BrdU detection, paraffin‐embedded 4 µm sections were deparaffinized, treated with HCl 2N for 1h and then with 0,1% trypsin at 37°C. Sections were sequentially incubated with goat serum (Abcam), mouse monoclonal anti‐BrdU antibody (Becton Dickinson, San Jose, CA) and with Dako EnVision+® System Labelled Polymer‐HRP anti‐mouse (Dako Corporation, Carpinteria, CA). Peroxidase binding sites were detected by Vector Novared Peroxidase (HRP) Substrate Kit (Vector Labs, Burlingame, CA). Harris haematoxylin solution (Sigma) was used to counterstain liver sections. Labelling index (LI) was expressed as the number of BrdU‐positive hepatocyte nuclei/100 nuclei. Mitotic index was calculated as the number of mitotic figures/1000 nuclei. Results were expressed as the means ± SD of 3 to 5 animals per group. At least 3000 hepatocyte nuclei per liver were scored.
6
Quantifying Hepatocyte Proliferation After High-Fat Diet
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To evaluate cell proliferation following HFD-induced hepatocyte injury, BrdU (1 mg⋅kg−1 body weight) was administered to all mice from week 17 to 19. After sacrifice, mice tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. All tissues were cut at a thickness of 4 μm using Microm HM315 Rotary microtome (Rankin Biomedical Corporation, Holly, MI, USA), deparaffinized, and immunostained for BrdU to identify proliferating cells, as previously described [23 (link)]. Mouse monoclonal anti-BrdU antibody (Becton Dickinson, San Jose, CA, USA) and HRP anti-mouse antibody (Dako Corporation, Carpinteria, CA, USA) were used as primary and secondary antibody, respectively. Harris hematoxylin solution (Sigma Aldrich) was used to counterstain tissue sections. Pictures were taken using Zeiss Axio Lab.A1 (Carl Zeiss Microscopy, B, Oberkochen, Germany). Labeling index (LI) was expressed as the number of BrdU-positive hepatocyte nuclei/100 in ≥50 fields at 40× enlargement. The results were expressed as the mean ± SD of 3 to 5 mice per group.
7
Quantifying Proliferating Cells in Lungs
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For labelling proliferating cells, 240 mg/kg 5-bromo-2′-deoxyuridine (Sigma Aldrich Corp.) was injected intraperitoneally into mice 1.5 h before lethal anesthesia. Lungs were fixed in BrdU fixing solution after removal. Five-micrometer paraffin sections were dewaxed and rehydrated. The peroxidase was quenched with methanol and H2O2 for 30 min. The slides were then incubated with HCl for 15 min, and blocked with milk powder for 30 min. BrdU immunostaining was performed using a mouse monoclonal anti-BrdU antibody (Becton Dickinson). The binding of BrdU was visualized by Dako Real Detection System (Agilent, Santa Clara, CA, USA) following the instructions of the manufacturer. The slides were scanned by PANNORAMIC 1000 slide digitalization system (3DHistech, Budapest, Hungary). One thousand nuclei per lung were counted on digital photographs using QuantCenter software (3DHistech, Budapest, Hungary).
8
Immunohistochemical Analysis of Cell Proliferation in Liver
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Liver sections were fixed in 10% of buffered formalin and processed for immunohistochemistry (IHC). For BrdU detection, paraffin‐embedded 4 µm sections were deparaffinized, treated with HCl 2N for 1 h and then with 0.1% trypsin at 37°C. Sections were sequentially incubated with goat serum (Abcam), mouse monoclonal anti‐BrdU antibody (Becton Dickinson) and with Dako EnVision+® System Labelled Polymer‐HRP anti‐mouse (Dako). Peroxidase binding sites were detected by Vector NovaRED Peroxidase (HRP) Substrate Kit (Vector Laboratories). Harris haematoxylin solution (Sigma‐Aldrich) was used to counterstain liver sections. Labelling index (L.I.) was expressed as the number of BrdU‐positive nuclei/field (at x40 magnification). Ten to 50 fields per liver were scored. A segment of the duodenum, an organ with a high rate of cell proliferation, was used as a positive control for BrdU incorporation.
9
Immunohistochemical Analysis of Embryonic Development
10
Cell Proliferation and Apoptosis Assays
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To examine cell proliferaton, mice were injected with 50 mg BrdU/kg 60 minute before to perfusion with a 3:1 70% EtOH:acetic acid. For anti-BrdU immunohistochemistry, brains were embedded in paraffin, sectioned on a microtome at 16um, and mounted on glass slides. Sections were deparaffinized, rehydrated, and treated with 1M HCl for 30 minutes at 37°C. Then, the slides were incubated with mouse anti-BrdU monoclonal antibody (1:200 dilution; BD Biosciences, Mississauga, ON, Canada) overnight followed by incubating in biotinylated horse anti-mouse immunoglobulin (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) for 1 hour on the next day. Lastly, the slides were stained using the VECTASTAIN Elite ABC kit (Vector Laboratories) and 3, 30-diaminobenzidine (SigmaeAldrich). For assessing cell death, the mice were processed for perfusion and the brains for sectioning as described for the cell proliferation work, above. The slides were immuno-stained with ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (S7111, ApopTag FITC-direct, Chemicon).
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