BrdU staining was performed by continuous intraperitoneal injection of BrdU (50 mg/kg) twice daily for 5 days before behavioral tests in advance. After the animals were deeply anesthetized and perfused with ice-cold 0.1% MPBS and 4% paraformaldehyde, the brain tissue was collected and dehydrated in a sucrose solution. Continuous coronal slices were cut into 35 μm slices using a frozen slicer. Hippocampal sections were incubated with 10% BSA (containing 0.3% Triton X-100) at room temperature for 1 h. The corresponding primary antibodies were added and incubated with the sections overnight at 4°C: mouse anti-BDNF (1:500, Abcam; ab203573), rat anti-BrdU (1:100, AbD Serotec; OBT0030), and mouse anti-Nestin (1:1000, Millipore, MAB5326). After washing with PBS, fluorescent secondary antibody (Invitrogen) was added and incubated at room temperature for 2 h without light. After washing with PBS, a DAPI staining solution was added, and the plates were sealed and observed under a confocal microscope (LSM510, Carl Zeiss, Goettingen, Germany). A total of 7–8 sections were randomly selected from each mouse for image acquisition and statistical analysis.
Obt0030
Manufactured by Merck Group
Sourced in United Kingdom
The OBT0030 is a piece of laboratory equipment designed for various applications within a research or clinical setting. It serves as a core function for conducting specific analyses or procedures. The detailed specifications and intended use of this product are not available in this response.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (1 mentions)
Mab5326, by Merck Group (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 anti rat, by Thermo Fisher Scientific (1 mentions)
Z0334, by Abcam (1 mentions)
Ab97959, by Merck Group (1 mentions)
Mab377, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Mab377, by Merck Group (1 mentions)
Mab353, by Merck Group (1 mentions)
Ab2253, by Merck Group (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Mab3034, by Merck Group (1 mentions)
Ab6946, by Abcam (1 mentions)
β agarase, by New England Biolabs (1 mentions)
5 protocols using obt0030
1
Analyzing Hippocampal Neurogenesis via BrdU Staining
2
Immunolabeling of Neurogenic Markers in Brain
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For the BrdU immuno-labeling, the brain sections were rinsed three times in 0.1 M PBS and incubated in 2 N HCl at 37°C for 15 min, followed by 0.1 M borate buffer (pH = 8.6) for 10 min and washed 3× with 0.1 M PBS. Sections were incubated in blocking solution for 40 min. Then, sections were incubated with some combinations of the following primary antibodies: rat anti-BrdU (1:500; AbD Serotec Cat # OBT0030, RRID:AB_609568 ), mouse anti-NeuN (1:500; Millipore Cat # MAB377, RRID:AB_2298772 ), rabbit anti-GFAP (1:100; Dako Cat # Z0334, RRID:AB_10013382 ), rabbit anti-Sox2 (1:500, Abcam Cat # AB97959; RRID:AB_2341193 ), guinea pig anti-doublecortin (DCX, 1:1000: Millipore Cat# AB2253, RRID:AB_1586992 ) in blocking solution at 4°C overnight. Sections were rinsed 3× with 0.1 M PBS, and incubated in 0.1 M PBS containing 10% fetal bovine serum and conjugated secondary antibodies (Alexa Fluor® 488 anti-rat Cat # A-21208; Alexa Fluor® 594 anti-rat Cat # A-11007; Alexa Fluor® 488 anti-mouse Cat # A32723; Alexa Fluor® 594 anti-rabbit; Alexa Fluor® 594 Cat# R37117; anti-guinea pig Cat# A-11076; dilution 1:1000; Thermo Fisher) for 1 h at room temperature and washed 3× with 0.1 M PBS. Nuclear counterstaining was done with 4′,6-diamidino-2-phenylindole (DAPI; Abcam Cat # ab104139, Cambridge, MA, USA).
3
Immunohistochemical Analysis of Neural Stem Cells
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For immunohistochemical processing, eight 35-µm-thick slices were randomly selected from each brain. Tissue samples were treated with 2 N HCl for 10 minutes at 37°C, followed by 0.1 M borate buffer at pH 8.5 for 10 minutes. Brain sections were rinsed four times in 0.1 M PBS and incubated in the blocking solution (PBS 0.1 M, Triton-X100 0.03% and 10% fetal bovine serum) for 50 minutes. Subsequently, the free-floating samples were incubated overnight with primary antibodies rat IgG anti-BrdU (Bromodeoxyuridine), a marker for cell proliferation (1:500; Bio-Rad, Kidlington, UK; Cat# OBT0030) and anti-Sox2, a marker for neural stem cells (1:500; Millipore, Billerica, MA, USA; Cat# AB5603) at 4ºC. Sections were then rinsed 4× with 0.1 M PBS and incubated with the same blocking solution containing the conjugated secondary antibodies (1:1000 Alexa Flour 488 anti-rat Cat# A-21208, and 1:1000 Alexa Flour 594 anti-rabbit Cat # R37117 Thermo-Fisher, Waltham, MA, USA) for 1 hour at room temperature. After rinsing (4× with 0.1 M PBS), nuclear staining was done with DAPI (Abcam Cambridge, MA, USA; Cat# ab104139). The sections were washed with 0.1 M PBS and mounted on glass slides and covered using Vectashield mounting media (Vector Laboratories, Burlingame, CA, USA; Cat# H-1000).
4
Immunohistochemical Analysis of Neurogenesis
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DNA was denatured with 2N HCl during 10 min at 37°C followed by 10 min in boric acid 0.1 M (pH 8.5) at room temperature. The tissues were rinsed in PBS-Triton-X-100 0.03% three times for 5 min, and blocked with goat serum 10%. They were incubated with primary antibodies for 20 h at 4°C: rat anti–BrdU (1:400; AbD Serotec Cat# OBT0030 RRID: AB609568) plus mouse anti-Nestin (1:400; Millipore Cat# MAB353 RRID:AB94911), guinea pig anti-DCX (1:500, Millipore Cat# AB2253 RRID:AB1586992), or mouse anti-NeuN (1:400; Merck Cat# MAB377 RRID:AB11210778). After rinsing, the sections were incubated with appropriate secondary antibodies (1:1000, Alexa Fluor 488 for BrdU, Alexa Fluor 594 for cell markers; Invitrogen, Life technologies) for 60 min at room temperature. The brain tissues were mounted with fluorescence solution Vecta Mount (Vector, H-5000) and quantified under a fluorescence microscope Axio-observer D2 (Zeiss, Germany).
5
DNA Fiber Analysis of Replication Dynamics
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As described previously (25 (link)), asynchronous DMS114 and H524 cells were sequentially labeled with 20 μmol/L IdU for 20 minutes and 50 μmol/L CldU for 20 minutes. To preserve long genomic DNA fibers, cells were embedded in low melting point agarose plugs and incubated in cell lysis buffer with proteinase K at 50°C overnight. Washed plugs with TE buffer, and then melted plugs in 0.1 mol/L MES (pH 6.5) at 70°C for 20 minutes. Agarose was subsequently degraded by adding 2 μL of β-agarase (New England Biolabs). DNA fibers were then stretched onto salinized coverslips (Genomic Vision, cov-002-RUO) using an in-house combing machine. Combed DNA on coverslips was then baked at 60°C for 2 hours and denatured in 0.5 N NaOH for 20 minutes. IdU, CldU, and ssDNA were detected using a mouse antibody directed against BrdU (IgG1, Becton Dickinson, 347580, 1:25 dilution), a rat antibody directed against BrdU (Accurate Chemical, OBT0030, 1:200 dilution), and a mouse antibody directed against ssDNA (IgG 2a, Millipore, MAB3034, 1:100), respectively. The secondary antibodies used were goat anti-mouse Cy3 (Abcam ab6946), goat anti-rat Cy5 (Abcam, ab6565), and goat anti-mouse BV480 (Jackson ImmunoResearch, 115-685-166) for ssDNA. Slides were scanned with a Fiber-Vision Automated Scanner (Genomic Vision). Replication signals on single DNA fibers were analyzed using FiberStudio (Genomic Vision).
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