Immunohistochemistry was used to evaluate NSC proliferation in SVZ and SGZ by BrdU staining. Firstly, the brain slices were incubated with 2 N HCl for 30 min to denaturate the DNA at 37°C. After being incubated with 0.1 mol L−1 boric acid (pH 8.5) for 10 min at room temperature followed by three times washing with 0.1 M PBS, the slices were blocked by 2% goat serum and 0.3% Triton X-100 for 2 h at room temperature, then incubated with the mouse monoclonal anti-BrdU antibody (1:200, Abcam, United Kingdom) at 4°C overnight. The next day, after three washings with 0.1 M PBS, the slices were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies for 2 h at room temperature. BrdU-positive cells were counted within defined regions of interest in the SVZ and SGZ. In total, the mean numbers of BrdU-positive cells of six brain slices for each rat, spaced approximately 200 μm apart, were examined by the observer blindly. For each slice, five regions were captured by fluorescence microscopy (BX51, Olympus, Tokyo, Japan), and the planar area enclosed by each region was 50 × 50 μm. The edges of the captured regions were defined according to structural details to ensure the fields did not overlap (Zhang et al., 2009 (link)). The density of positive cells was presented as the total number of BrdU-positive cells in the SVZ and SGZ.
Mouse monoclonal anti brdu antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Mouse monoclonal anti-BrdU antibody is a primary antibody that recognizes bromodeoxyuridine (BrdU), a synthetic nucleoside analog of thymidine. It can be used to detect and quantify cell proliferation in various applications.
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Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (3 mentions)
Fluorescein isothiocyanate conjugated rabbit anti mouse immunoglobulin antibody, by Jackson ImmunoResearch (1 mentions)
α sma, by Merck Group (1 mentions)
Eclipse ti u fluorescence microscope, by Nikon (1 mentions)
Bx51 fluorescence microscope, by Nikon (1 mentions)
Axiovision, by Zeiss (1 mentions)
Ab80633, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Rabbit anti gfap polyclonal antibody, by Abcam (1 mentions)
Dapi fluoromount g, by Beyotime (1 mentions)
8 protocols using mouse monoclonal anti brdu antibody
1
Quantifying Neural Stem Cell Proliferation
2
BrdU Incorporation Assay for Evaluating Cell Proliferation
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The treated or untreated cells that had been transfected with miR-505 mimics, anti-miR-505, or negative controls were rinsed with RPMI-1640 medium and then cultured in fresh medium with 10 µM bromodeoxyuridine (BrdU; Sigma-Aldrich Co) at room temperature for ~1 hour. After washing in cold PBS, the cells were then fixed in 70% ice-cold ethanol. Then, the cells were resuspended in 2 mol/L HCl at 37°C for 30 minutes, after which washing with PBS and hybridization using a mouse monoclonal anti-BrdU antibody (Abcam) at a dilution of 1:100 in PBST (PBS containing 0.1% BSA and 0.2% Tween-20, pH 7.4) at 4°C overnight took place. The next day, the cells were rinsed with PBST and then incubated with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin antibody (Jackson Immuno Research, Lancaster, PA, USA) at room temperature for approximately 2 hours. Then, the cells were stained with DAPI for around 10 minutes and then photographed.
3
Quantifying Cell Proliferation via BrdU Assay
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Untreated cells or cells transfected with plasmids were washed thoroughly with medium and cultured in fresh medium containing 10 μM bromodeoxyuridine (BrdU; Sigma‐Aldrich) for 1 hour. Cells were then either harvested directly or left to grow in BrdU‐free medium for varying lengths of time prior to being harvested.
The resulting cell pellets were washed with PBS. Cells were then hybridized with a mouse monoclonal anti‐BrdU antibody (Abcam, USA), diluted at a ratio 1:100 in PBST and incubated overnight at 40°C. After rinsing with PBST, cells were incubated with FITC‐conjugated rabbit anti‐mouse immunoglobulin antibody (Immuno Jackson, USA) diluted at a ratio1:400 in PBST. After 2 hour incubation at room temperature in the dark, cells were washed with PBS and stained with DAPI solution for 10 minutes before being photographed.
The resulting cell pellets were washed with PBS. Cells were then hybridized with a mouse monoclonal anti‐BrdU antibody (Abcam, USA), diluted at a ratio 1:100 in PBST and incubated overnight at 40°C. After rinsing with PBST, cells were incubated with FITC‐conjugated rabbit anti‐mouse immunoglobulin antibody (Immuno Jackson, USA) diluted at a ratio1:400 in PBST. After 2 hour incubation at room temperature in the dark, cells were washed with PBS and stained with DAPI solution for 10 minutes before being photographed.
4
PDGF and Fibronectin Regulate SMC Proliferation
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Human and mouse SMCs were serum-starved for 48 hours to induce quiescence. Cells were washed and stimulated with PDGF-BB (20 ng/mL), cFn (20 μg/mL) or pFn (20 μg/mL), and recombinant EDA-containing or EDA-lacking peptides (10 μg/mL) for 24 hours. Cells were incubated with 10 μM BrdU for the last 12 hours of treatment (50 (link)). Carotid artery sections from BrdU-treated mice or cultured SMCs were fixed with 4% PFA (in PBS). DNA hydrolysis was performed by treating the cells/sections with 2 M HCl for 20 minutes. Samples were subjected to immunofluorescence staining with a mouse monoclonal anti-BrdU antibody (1:200, Abcam) together with αSMA (1:200, MilliporeSigma) for 3 hours at 37°C and labeled with appropriate Alexa Fluor 488 and Alexa Fluor 546 secondary antibodies (1:400, Abcam). Nuclei were stained with Hoechst (5 μg/mL) before mounting. Images of the sections were acquired using an Olympus BX51 fluorescence microscope, and images of cells were acquired using the Nikon Eclipse Ti-U fluorescence microscope. The number of BrdU-positive cells in the neointima or the media and the total number of nuclei were determined using ImageJ software. The percentage of BrdU-positive cells was calculated as (number of BrdU-positive nuclei in the neointima or media/total number of nuclei) × 100.
5
Immunohistochemistry and BrdU Analysis
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Tissues were separated after the sacrifice of rats, and fixed with 10% formalin for 24 h. OCT compound was used for tissue embedding, and 10-μm thickness tissues were achieved using a frozen microtome. Mouse anti-P21 antibody (ab80633, Abcam, Cambridge, UK) and mouse anti-PCNA antibody (ab29, Abcam, Cambridge, UK) were used in this study. Then, incubation with biotinylated secondary antibody and visualization with Vectastain Elite ABC HRP kit. Zeiss AxioVision was applied for visualizing.
For the BrdU staining, the BrdU was firstly dissolved into 10 mg/mL using PBS. The animals were treated with BrdU (100 mg/kg) through intraperitoneal injection. Tissue separation, embedding, section, and fixation were conducted as described above. Then, the sections were incubated with a mouse monoclonal anti-BrdU antibody (Abcam, Cambridge, UK). The proliferation data of tissues were analyzed by counting the BrdU stained positive cells (nuclei).
For the BrdU staining, the BrdU was firstly dissolved into 10 mg/mL using PBS. The animals were treated with BrdU (100 mg/kg) through intraperitoneal injection. Tissue separation, embedding, section, and fixation were conducted as described above. Then, the sections were incubated with a mouse monoclonal anti-BrdU antibody (Abcam, Cambridge, UK). The proliferation data of tissues were analyzed by counting the BrdU stained positive cells (nuclei).
6
BrdU Incorporation Assay for Cell Proliferation
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Untreated cells or cells transfected with siRNA or plasmid were washed thoroughly with medium and cultured in fresh medium containing 10 µM bromodeoxyuridine (BrdU; Sigma-Aldrich) at 37 °C for 1 h. Then the cells were washed with PBS, fixed in 70% ice-cold ethanol. Fixed cells were treated with 2N HCl and incubated for 30 min at RT. After washing with PBS, cells were hybridized with a mouse monoclonal anti-BrdU antibody (dilution 1:100, Abcam, USA) overnight at 4 °C. Cells were then rinsed with PBST and incubated with FITC-conjugated rabbit anti-mouse immunoglobulin antibody (Jackson Immuno Research, Lancaster, PA, USA) diluted at a ratio 1:400 in PBST. After 2 h incubation at RT in the dark, cells were washed with PBS and stained with DAPI solution for 10 min before taking photos.
7
Immunofluorescent Staining for BrdU Detection
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The glass slides were heated and then immersed in order in xylazine, alcohol 100%, 95%, 70%, 30%, PBS, Triton X-100 at room temperature (RT), followed by immersion in HCl 2 molar at 37°C and sodium tetraborate buffer at RT. To reduce nonspecific protein bindings, the slides were incubated with blocking solution (10% normal goat serum and 0.1% bovine serum albumin in PBS) at RT for 30 min. Then, slides were incubated with mouse anti-BrdU monoclonal antibody (abcam, UK) –1:10 dilution in 10% normal goat serum, 0.1% bovine serum albumin and 1% glycine in PBS– overnight at 4 °C. After three times washing with PBS, sections were incubated with goat anti-mouse FITC-labeled secondary antibody (abcam, UK, 1:200 in dilution buffer) at RT for two hours. To stain the nuclei for calculating the total cell numbers, sections were incubated with DAPI (10µg/ml, Sigma) for 7 min at RT. Then, the cover slides were mounted with Entalan (Merck, Germany).
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The glass slides were heated and then immersed in order in xylazine, alcohol 100%, 95%, 70%, 30%, PBS, Triton X-100 at room temperature (RT), followed by immersion in HCl 2 molar at 37°C and sodium tetraborate buffer at RT. To reduce nonspecific protein bindings, the slides were incubated with blocking solution (10% normal goat serum and 0.1% bovine serum albumin in PBS) at RT for 30 min. Then, slides were incubated with mouse anti-BrdU monoclonal antibody (abcam, UK) –1:10 dilution in 10% normal goat serum, 0.1% bovine serum albumin and 1% glycine in PBS– overnight at 4 °C. After three times washing with PBS, sections were incubated with goat anti-mouse FITC-labeled secondary antibody (abcam, UK, 1:200 in dilution buffer) at RT for two hours. To stain the nuclei for calculating the total cell numbers, sections were incubated with DAPI (10µg/ml, Sigma) for 7 min at RT. Then, the cover slides were mounted with Entalan (Merck, Germany).
8
Immunohistochemical Analysis of Neuroinflammation
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After fixation (Sect. 4.8), brain tissues were dehydrated in 30% sucrose for 72 h, sliced in the coronal plane at 30 μm thickness using a freezing microtome, and processed for BrdU staining. First, genomic DNA in slices was denatured by treatment with 2 Ν HCl for 30 min at 37 °C. Slices were then neutralized by immersed in 0.1 M sodium borate for 10 min, and then rinsed with PBS. The tissues were permeabilized with 0.3% Triton X-100 PBS solution, blocked with 10% goat serum for 60 min at r/t, and incubated with mouse anti-IBA-1 monoclonal antibody (1:1000, Abcam, UK), rabbit anti-GFAP polyclonal antibody (1:1000, Abcam), and mouse anti-BrdU monoclonal antibody (1:1000, Abcam) overnight at 4 °C. Slices were subsequently rinsed with PBS, incubated with FITC-conjugated goat anti-rabbit IgG Alexa Fluor 594 conjugate (1:500, Abcam, UK) and goat anti-mouse IgG Alexa Fluor 488 conjugate (1:500, Abcam) for 2 h at r/t, counterstained and mounted with DAPI Fluoromount-G (Beyotime), and imaged using an IX71 fluorescence microscope (Olympus, Tokyo, Japan).
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