Three‐dimensional cultures were fixed with ice‐cold 4% paraformaldehyde in PBS at 4°C for 30 min. After permeabilization with 0.3% Triton X‐100 in PBS for 10 min., the cells were blocked with 5% normal goat serum in PBS for 1 hr at room temperature. Then, the cells were incubated with primary anti‐E‐cadherin (36/E‐cadherin; BD Biosciences, San Jose, CA, USA) and anti‐surfactant protein‐C pro‐peptide (pro‐SP‐C; AB3786; Chemicon International, Temecula, CA, USA) and anti‐vimentin (Clone V9; DAKO, Glostrup, Denmark) antibodies at 4°C overnight. After washing with PBS, the cells were incubated with Alexa 488‐conjugated IgG (1:600; Invitrogen, Carlsbad, CA, USA) as secondary antibodies and the nuclear dye propidium iodide for 30 min. at room temperature, and observed as described previously 16 . For a 5‐bromo‐2′‐deoxyuridine (BrdU) assay, HBE135 cell colonies cultured for 9 days were incubated for a further 3 days with 10 μM BrdU (Nacalai Tesque, Kyoto, Japan). The colonies were fixed in 4% paraformaldehyde, and BrdU incorporation was assayed by immunofluorescence using an anti‐BrdU antibody (OBT0030; AbD Serotec, Oxford, UK).
Bromodeoxyuridine (brdu)
Manufactured by Nacalai Tesque
Sourced in Japan
BrdU is a synthetic thymidine analog that is commonly used in cell proliferation assays to detect DNA synthesis and cell division. It can be incorporated into the newly synthesized DNA of dividing cells, allowing for the identification and quantification of proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Agilent Technologies (2 mentions)
Biorevo software, by Keyence (2 mentions)
Bz 9000 microscope, by Keyence (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by Abcam (1 mentions)
Fluoview fv1000 d confocal laser scanning microscope, by Olympus (1 mentions)
Bromodeoxyuridine (brdu), by Bio-Rad (1 mentions)
Anti brdu antibody, by Bio-Rad (1 mentions)
Obt0030, by Bio-Rad (1 mentions)
Anti vimentin clone v9, by Agilent Technologies (1 mentions)
Anti e cadherin 36 e cadherin, by BD (1 mentions)
Alexa fluor 488 and 555 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Diaminobenzidine substrate kit, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Bromodeoxyuridine (brdu), by Roche (1 mentions)
Bromodeoxyuridine (brdu), by Olympus (1 mentions)
9 protocols using bromodeoxyuridine (brdu)
1
Immunofluorescence Staining of 3D Cultures
2
Evaluating Endogenous Neurogenesis in SVZ and DG
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In order to evaluate the endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus, 5-bromo2′-deoxyuridine (BrdU, NACALAI TESQUE INC., Kyoto, Japan) was injected into all rats at a concentration of 50 mg/kg body weight, with five consecutive intraperitoneal injections every 12 h from day 13 to 15 after MCAO before euthanasia to label proliferative cells [44 (link), 45 (link)].
3
Islet-Myoblast Co-Transplantation Dynamics
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Before the procedure, recipient mice were randomly divided into the following two groups: islet alone group (n = 5) and islet + myoblast group (n = 5). Islets were transplanted under the left kidney capsule using the method described above. Bromodeoxyuridine (BrdU) (Nacalai Tesque) (50 mg/kg body weight) was intraperitoneally administered for four consecutive days, starting on the third day after transplantation. At 4 h after the last BrdU injection, the kidneys were dissected. We performed immunostaining of insulin and BrdU using the appropriate antibodies. The proportion of insulin and BrdU double-positive cells among the total insulin-positive cells was calculated.
4
Labeling Dividing Cells during Mitosis
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5
Quantifying Pancreatic Beta Cell Proliferation
6
Quantifying Pancreatic Beta-cell Proliferation
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Mice were intraperitoneally injected with BrdU (100 µg/g; Nacalai Tesque, Inc) for 3 days and sacrificed. Three pancreatic tissue sections (100 µm apart) from each animal were prepared by fixation and paraffin-embedding. The sections were immunostained with antibodies to insulin, muscarinic acetylcholine receptor M1 (Santa Cruz), BrdU (Dako), and muscarinic acetylcholine receptor M3 (Bioss). Biotinylated secondary antibodies, a Vectastain elite ABC kit and a diaminobenzidine substrate kit (Vector) were used to examine the sections under bright-field microscopy to determine the β-cell mass. Alexa Fluor 488- and 555-conjugated secondary antibodies (Invitrogen) were used for fluorescence microscopy analysis. All images were captured using a BZ-9000 microscope (Keyence). The percentage area of the pancreatic tissues occupied by β cells was calculated using BIOREVO software (Keyence)31 (link). In the BrdU staining experiment, 22–78 islets in each mouse were analysed to assess the frequency of BrdU-positive cells among the insulin-positive cells.
7
BrdU Labeling of Proliferative Cells in Mice
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WT and M3−/− mice were injected intraperitoneally with BrdU (Nacalai Tesque) at 50 mg/kg body weight as a marker of proliferation, and then euthanized at 3, 6, 15, and 24 h post-injection. After immunostaining, BrdU labeling was detected using the BrdU detection and labeling kit II (Boehringer-Mannheim) according to the manufacturer’s instructions. Following immunohistochemical staining for BrdU, the number of BrdU-positive cells was counted from at least 30 crypts of WT and M3−/− mice per time point.
8
BrdU-Based Quantification of Cell Proliferation
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To determine the number of newly generated cells, BrdU (10 mg/ml; Nacalai Tesque, Kyoto, Japan), a marker of cell proliferation, was administered intraperitoneally (100 μg/g of body weight dissolved in PBS). Six hours after injection, BrdU-labeled cells were detected as described previously (Takahashi et al., 2016 (link)). For quantification, the number of BrdU+ cells within the CVP was measured in Z-stack images acquired with a confocal laser microscope (FV1000-D). Signals larger than 10 μm2 were counted as BrdU+ nuclei. In the same images, the CVP area was measured using Olympus Fluoview software (Olympus), and the density of BrdU+ cells was calculated.
9
Quantification of Pancreatic Cell Proliferation
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Mice were intraperitoneally administered BrdU (Nacalai Tesque; 50 mg/kg body weight) for 4 consecutive days. Four hours after the last injection, the pancreas was dissected. We performed immunostaining insulin and BrdU with the antibodies as described above, and the proportion of insulin and BrdU double-positive cells to the total insulin-positive cells was calculated.
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