Porcine kidney 15 (PK15) cells purchased from ATCC (CCL-33) were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Scientific HyClone, Beijing, China), and incubated at 37 °C in a 5% CO2 atmosphere incubator. The PCV2 strains (GenBank No. EU366323) used in this study were isolated and purified previously by our team and stocked in our laboratory, the UV-inactivation was performed by UV radiation of the virus for 45 min in the hood. The anti-PCV2 Cap primary antibodies were produced by our team [12 (link), 13 (link)]. The primary monoclonal rabbit antibodies of p53, p21 and anti-BrdU were purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA). CDK2, Cyclin A and Cyclin E antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California, CA, USA). The monoclonal antibody of β-actin was purchased from sigma (Sigma-Aldrich, St. Louis, MO, USA). The FITC goat anti-mouse IgG was purchased from BD Biosciences (BD, San Jose, CA, USA).
Anti brdu
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-BrdU is a primary antibody that specifically recognizes bromodeoxyuridine (BrdU), a synthetic nucleoside analog of thymidine. This antibody can be used to detect and quantify cellular proliferation in various applications, such as immunohistochemistry and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Fitc goat anti mouse igg, by BD (1 mentions)
Cyclin e, by Santa Cruz Biotechnology (1 mentions)
Anti vdac1, by Proteintech (1 mentions)
Anti senp1, by Cell Signaling Technology (1 mentions)
Anti sumo1, by Cell Signaling Technology (1 mentions)
Anti flag tag, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Proteintech (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Anti ubiquitin, by Cell Signaling Technology (1 mentions)
Anti ubc9, by Cell Signaling Technology (1 mentions)
Anti phospho threonine, by Cell Signaling Technology (1 mentions)
Anti sumo2 3, by Cell Signaling Technology (1 mentions)
Anti senp2, by Abcam (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Anti ha tag, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
12 protocols using anti brdu
1
Porcine Kidney Cell Culture and PCV2 Infection
2
Western Blot Protein Analysis Protocol
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Cells were lysed in RIPA lysis buffer and the protein concentration of the cell lysates determined by BCA Protein Assay (Thermo Scientific, 23227). Equal amounts of protein were loaded onto SDS-PAGE gel and transferred to PVDF membranes. Western blotting was performed using primary antibodies and secondary antibodies conjugated with HRP. For immunoblotting, the following antibodies were used: anti-SENP2 (Abcam, ab58418, 1:1000), anti-HIF-1α (Cell Signaling Technology, 79233, 1:1000), anti-phospho-threonine (Cell Signaling Technology, 9386, 1:1000), anti-Ubc9 (Cell Signaling Technology, 4786, 1:1000), anti-BrdU (Cell Signaling Technology, 5292, 1:1000), anti-ubiquitin (Cell Signaling Technology, 3936, 1:1000), anti-cleaved-caspase 3 (Cell Signaling Technology, 9661, 1:1000), anti-SUMO1 (Cell Signaling Technology, 4930, 1:1000), anti-SUMO2/3 (Cell Signaling Technology, 4971, 1:1000), anti-SENP1 (Cell Signaling Technology, 11929, 1:1000), anti-SENP3 (Cell Signaling Technology, 5591, 1:1000), anti-HA-Tag (Cell Signaling Technology, 3724, 1:2000), anti-Flag-Tag (Cell Signaling Technology, 14793, 1:2000), anti-hexokinase 1 (Proteintech, 19662-1-AP, 1:1000), anti-hexokinase 2 (Proteintech, 22029-1-AP, 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:1000), anti-alpha tubulin (Proteintech, 66031-1-Ig, 1:5000), anti-GAPDH (Proteintech, 10494-1-AP, 1:5000).
3
Antibody Panel for Cellular Signaling
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The following antibodies were used in this study: anti-PGAM1 (NBP1-49532; Novus Biologicals), anti-CtIP (sc-271339; Santa Cruz Biotechnology, Inc.), anti-Mre11 (ab214; Abcam), anti–H2AX-pS139 (9718; Cell Signaling Technology), anti–β-actin (60008-1-Ig; Proteintech), anti–Lamin B1 (12987-1-AP; Proteintech), anti-GAPDH (60004-1-Ig; Proteintech), anti-RPA32 (ab2175; Abcam), anti–RPA32-pS4S8 (NBP1-23017; Novus Biologicals), anti-BrdU (5292; Cell Signaling Technology), anti-Cdh1 (ab3242; Abcam), anti-p21 (2947; Cell Signaling Technology), anti-RAD51 (sc-8349; Santa Cruz Biotechnology, Inc.), anti-PGD (sc-398977; Santa Cruz Biotechnology, Inc.), anti-PHGDH (ab57030; Abcam), anti-IgG (2729; Cell Signaling Technology), anti-Histone H3 (4620; Cell Signaling Technology), anti-p53 (9282; Cell Signaling Technology), anti-p73 (ab202474; Abcam), and anti–Cleaved Caspase-3 (9661; Cell Signaling Technology).
4
Evaluating Renal Cell Carcinoma Cells
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All cell lines were STR (short tandem repeat) verified and mycoplasma tested. RCC4, RCC4 VHL (Sigma); RCC10, RCC10 VHL (gift of Dr Amato Giaccia); and 786-O, 786-O VHL (gift of Dr Sandra Turcotte) cells were maintained in high-glucose DMEM medium supplemented with 10% bovine calf serum at 37 °C and 5% CO2. For hypoxia incubation, a 1% O2 environment was generated in a Ruskinn InVivo2 Hypoxia chamber. Lipofectamine 2000 (Life Technologies) was used for transfections. Crystal violet staining was performed with 0.05% crystal violet in 1% formaldehyde and 1% methanol. BrdU labeling was performed by supplementing cells with 0.03 mg/ml for 24 h, and staining with anti-BrdU (Cell Signaling) followed by Texas red-conjugated goat anti-mouse IgG. Non-specific FITC fluorescence was used as a background cell stain.
5
Immunofluorescence Microscopy of Cellular Targets
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Immunofluorescence was performed on cells cultured on glass‐bottom dishes (P35G‐1.5‐20‐C, MatTek, Ashland, MA, USA), as described previously.7 Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20℃, followed by three 5‐min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 h, followed by incubation with primary antibodies at 4℃ overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (D1306, Life Technologies, Carlsbad, CA, USA). Confocal microscopy was performed with the Ultraview Vox spinning‐disk confocal system (PerkinElmer) equipped with a Yokogawa CSU‐X1 spinning disk head and an electron‐multiplying charge‐coupled device camera (Hamamatsu C9100‐13) coupled to a Nikon Ti‐E microsope; image analysis was done using Volocity software (PerkinElmer). The following antibodies were used for immunofluorescence: anti‐phospho‐mTOR (Ser2448) (5536, Cell Signaling, Beverly, MA, USA), anti‐LAMP1 (555798, BD Biosciences, San Jose, CA, USA), anti‐BrdU (5292, Cell Signaling, Beverly, MA, USA), Alexa Fluor 568 goat anti‐mouse secondary (A‐11031, Life Technologies, Carlsbad, CA, USA), and Alexa Fluor 488 goat anti‐rabbit secondary (A‐11034, Life Technologies, Carlsbad, CA, USA).
6
Paraffin-Embedded Tissue Immunohistochemistry
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Tissue was embedded in paraffin, and 6-μm sections were cut, stained with either H&E or toluidine blue, and incubated overnight at 4°C with the following antibodies: anti-S100β (Dako, Carpinteria, CA), Ki67 (Novocastra Leica Microsystems, Buffalo Grove, IL), anti-BrdU, or anti-CC3 (Cell Signaling Technology, Danvers, MA). Visualization methods were as described (6 (link)).
7
Antibody Immunostaining Protocol
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All antibodies were purchased as follows: anti-NEDL2 (ab92711, Abcam), anti-Ret (ab134100, Abcam), anti-Neurofilament (ab50284, Abcam), anti-Akt (sc-8312, Sata Cruze), anti-pAkt (#4060, Cell Signaling), anti-S6K1 (#9202, Cell Signaling), anti-pS6K1 (#9234, Cell Signaling), anti-p85 (#4257, Cell Signaling), anti-p110 (#4249, Cell Signaling), anti-BrdU (#5259, Cell Signaling), anti-Myc (MBL), anti-Flag (MBL), anti-Hsp90 (sc-101494, Santa Cruz).
8
Evaluating siRNA Impacts on MCF7 Cell Proliferation
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After 72h of siRNA-1 and siRNA-CN treatment, MCF7 cells were exposed to 5-bromo 2-deoxyuridine (BrdU) (B5002, Sigma Aldrich) for 2h and fixed with cold ethanol. Cells were stained with anti-BrdU (5252, Cell signaling) followed by anti-mouse AlexaFluor-555 (#4409, Cell Signaling) and were then counterstained with blue fluorescent 4-6-diamidino-2-phenylindole (DAPI) for 10 min. Images for each treatment were taken from at least four different fields of the duplicates. Proliferation rate was calculated manually and blindly as the ratio of BrdU-positive nuclei (red) to total cell count (DAPI-positive nuclei, blue). Images were obtained using a fluorescent microscope (Zeiss-Axioimager A1 Carl, Germany).
9
BrdU Incorporation Assay for Proliferation
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2.5 × 105 MCF10A and 3.5 × 105 MDA MB 231 and MCF7
cells/well were grown on coverslips on a 6-well plate for 48 h. For
the BrdU assay, cells were incubated with 20 μM bromodeoxyuridine
(BrdU) for 4 h. Cells were gently washed with PBS 1X and fixed with
1 mL of 4% Paraformaldehyde (PFA) in PBS 1X for 20 min at room temperature.
Then the cells were permeabilized with 0.1% TritonX-100/PBS for 15
min and blocked with 5% BSA in 0.1% TritonX-100/PBS for 30 min at
room temperature. Anti-BrdU (Cell Signaling Technology, 5292, 1:200)
for BrdU, anti-V5 (CST, mAB13202, 1:1000) for V5 tag, and DAPI (Sigma,
D9542, 1:500) for nucleus staining were used. Afterward, the cells
were rinsed with PBS 1X (3 times, 10 min each) and mounted on Ibidi
Mounting Medium (IMM, Cat. 50001). Fluorescent images were captured
by using Olympus IX83 fluorescent microscope.
cells/well were grown on coverslips on a 6-well plate for 48 h. For
the BrdU assay, cells were incubated with 20 μM bromodeoxyuridine
(BrdU) for 4 h. Cells were gently washed with PBS 1X and fixed with
1 mL of 4% Paraformaldehyde (PFA) in PBS 1X for 20 min at room temperature.
Then the cells were permeabilized with 0.1% TritonX-100/PBS for 15
min and blocked with 5% BSA in 0.1% TritonX-100/PBS for 30 min at
room temperature. Anti-BrdU (Cell Signaling Technology, 5292, 1:200)
for BrdU, anti-V5 (CST, mAB13202, 1:1000) for V5 tag, and DAPI (Sigma,
D9542, 1:500) for nucleus staining were used. Afterward, the cells
were rinsed with PBS 1X (3 times, 10 min each) and mounted on Ibidi
Mounting Medium (IMM, Cat. 50001). Fluorescent images were captured
by using Olympus IX83 fluorescent microscope.
10
Comprehensive Antibody Protocol for Cellular Analysis
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