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Prb 452c

Manufactured by Fortrea
Sourced in United Kingdom

The PRB-452C is a laboratory instrument designed for the measurement and analysis of various physical and chemical properties. It features a compact and durable construction, providing reliable performance in a wide range of laboratory settings. The core function of the PRB-452C is to accurately capture and process data related to the parameter or sample under investigation, without making claims about its intended use or applications.

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4 protocols using prb 452c

1

Cell Fixation Protocols for Mitotic Spindle Proteins

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All cell fixation procedures were done at room temperature (∼22°C). For Mad2 staining in Figs. 2, 4, and S3, cells were fixed in PBS + 3.7% formaldehyde with 0.5% Triton X-100 for 10 min. For BubR1staining in Fig. 3 A, cells were preextracted in PBS + 0.5% Triton X-100 for 1 min, then fixed in PBS + 3.7% formaldehyde and 0.5% Triton X-100 for 10 min. For Mps1 staining in Fig. 3 B, cells were prefixed for 5 s in PBS + 3.7% formaldehyde, extracted in PBS + 0.5% Triton X-100 for 1 min, then fixed in PBS + 3.7% formaldehyde with 0.5% Triton X-100 for 10 min. The following primary antibodies were used: rabbit polyclonal anti-Mad2 (1:500; PRB-452C; Covance), mouse monoclonal anti-Hec1 9G3 (1:1,000; ab3613; Abcam), rabbit anti-BubR1 polyclonal (1:1,000; a gift from W. Dai, New York Medical College, Valhalla, NY), rabbit anti–CENP-C polyclonal (1:1,000; a gift from B. Black, University of Pennsylvania, Philadelphia, PA), and mouse anti-Mps1 monoclonal NT (1:100; 05-682; EMD Millipore).
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2

Immunofluorescence Staining of Mitotic Proteins

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The following primary antibodies were used for immunofluorescence: ACA (anticentromere antibody; Geisel School of Medicine; 1:1000), Astrin (C-terminal; 1:1000) (Mack and Compton, 2001) (link), CENP-A (#2186; Cell Signaling; 1:500), Hec1 (C-11; Santa Cruz; 8 ug/mL), HURP (gift from Erich Nigg; 1:1000) (Silljé et al., 2006) (link), Mad2 (PRB-452C; Covance; 1:500), pHec1 S44 (gift from Jennifer DeLuca; 1:500), Ska3 (gift from Erich Nigg; 1:1000) (Gaitanos et al., 2009) (link). Secondary antibodies used were Alexa Fluor® 488, 568, 594, and 647 (Molecular Probes; 1:2000) . DAPI (Molecular Probes) was used at 400 ng/mL.
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3

Immunoblotting and Immunofluorescence of Mitotic Spindle Proteins

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The following antibodies were used for immunoblot (IB) and immunofluorescence (IF) experiments: Human anti‐ACA (1:30, Antibodies Incorporated #15‐234), Mouse anti α‐Tubulin Alexa Fluor 488 conjugated (IF:1:100, Life Technologies #322588), Rabbit anti‐ MAD2 (IF:1:1000, IB: 1:500; Biolegend #924601 or previously Covance #PRB‐452C)), sheep anti‐BUB1 (1:100, gift from Dr. S. Taylor), rabbit anti‐MPS1 (1:100, gift from Dr. H. Yu), rabbit anti‐Securin (IB: 1:500, Invitrogen #700791) rabbit anti‐ZW10 (IF:1:100; IB:1:500, Abcam #ab21582), rabbit anti‐HEC1 (1:100; gift from Dr. R. Benezra), rabbit anti α‐Tubulin WB:1:500 Cell Signaling Technology #11H10). The following secondary antibodies were used at 1:200 for IF experiments: goat‐anti‐human‐Alexa‐633 (Life Technologies #A21091), donkey‐anti‐rabbit‐Alexa‐568 (Life Technologies #A10042) and Cy5 secondary antibody pre‐absorbed against goat serum proteins (Jackson Immunoresearch). Monastrol (Sigma #M8515), and nocodazole (Sigma #M1404) were dissolved in dimethyl sulfoxide (DMSO) (Sigma) and added to the CZB culture media at final concentrations of 100 μM and 5 μM, respectively. N‐Acetyl‐L‐Cysteine (NAC) (Abcam #Ab143032), ROS inhibitor, was dissolved in embryo water and added to CZB at final concentration of 5 mM. In vitro maturation of drug‐treated oocytes was performed in organ culture dishes.
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4

Imaging Meiosis I Oocyte Centromeres

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Meiosis I oocytes, at 5 h after release from IBMX, unless otherwise stated, were fixed and permeabilized in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl2) containing 4% paraformaldehyde and 0.5% Triton X-100 and then labeled with calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) serum, a human centromere antiserum, (1:300; a gift from G. Fitzharris, University College London, London, England, UK), rabbit anti-Mad2 (PRB-452C; 1:300; Covance), mouse anti–β-tubulin (T4026; 1:1,000; Sigma-Aldrich), and 10 µg/ml Hoechst 33342 (Sigma-Aldrich). Serial z sections of fixed oocytes in PBS were acquired at room temperature using a Plan Apochromat 63×, 1.4 NA oil differential interference contrast objective and a laser-scanning confocal microscope imaging system (LSM 510 META; Carl Zeiss) with the following band pass emission filters in nm 385–470 (Hoechst 33342), 505–530 (Alexa Fluor 488), and 585–615 (Alexa Fluor 546). Z sections were analyzed and projected into one picture using the LSM image browser (Carl Zeiss). The images were then assembled by Illustrator CS2 (Adobe).
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