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).
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.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
Cenp a, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Monastrol, by Merck Group (1 mentions)
Ab21582, by Abcam (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Cy5 secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit anti α tubulin, by Cell Signaling Technology (1 mentions)
Donkey anti rabbit alexa 568, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Illustrator cs2, by Adobe (1 mentions)
Lsm image browser, by Zeiss (1 mentions)
Mouse anti β tubulin t4026, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
4 protocols using prb 452c
1
Cell Fixation Protocols for Mitotic Spindle Proteins
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.
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.
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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