Paraffin sections were deparaffinized and rehydrated by immersion in xylene followed by a graded series of ethanols as previously described.14 (link) Antigen retrieval was performed by using a heat-induced method with citrate buffer (Dako). After blocking with 10% normal donkey serum and goat anti-mouse IgG (Sigma-Aldrich) for 1 hour, sections were incubated with a primary antibody at 37°C for 1 hour, followed by the application of corresponding secondary antibody (Alexa dyes; Invitrogen) at 37°C for 1 hour. Omission of the primary antibodies on parallel sections was used as negative control. The following primary antibodies were used c-Kit (goat polyclonal; R&D systems), CD45 (rabbit polyclonal; Abcam), serine-10 phosphorylated histone-H3 (HP3; rabbit polyclonal; Abcam), anti-BrdU (biotinylated mouse monoclonal; Abcam), Aurora B kinase (Rabbit polyclonal, Abcam), activated caspase 3 (mouse monoclonal; BD), and tropomyosin (mouse monoclonal; Abcam). Nuclei were counterstained by incubation for 5 minutes with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). The total number of positively stained cells was quantified per zone observed and corrected per area of corresponding zone (mm2). All images were obtained with a fluorescent microscope (Olympus IX81; Olympus America Inc, Center Valley, PA) or a confocal laser-scanning module (LSM710; Carl Zeiss MicroImaging).
Aurora b kinase
Manufactured by Abcam
Aurora B kinase is a serine/threonine protein kinase that is a key regulator of mitosis. It is involved in chromosome segregation and cytokinesis during cell division.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (1 mentions)
Alexa dye, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
Citrate buffer, by Agilent Technologies (1 mentions)
Activated caspase 3, by BD (1 mentions)
Anti brdu, by Abcam (1 mentions)
Nbt bcip substrate, by Promega (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Cenp a, by Cell Signaling Technology (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Anti α mhc, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti sarcomeric α actinin, by Merck Group (1 mentions)
Anti vimentin, by Santa Cruz Biotechnology (1 mentions)
3 protocols using aurora b kinase
1
Immunohistochemical Staining of Tissue Sections
2
Western Blot Analysis of Cell Signaling
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Cell lysates were prepared in RIPA lysis buffer (Santa Cruz Biotech, Santa Cruz, CA), separated by NuPage gel (Invitrogen) and blotted onto polyvinylidenedifluoride membranes. Membranes were blocked with 5% BSA in TBS-T and incubated with primary (1:1,000) and secondary (1:5,000) antibodies. Primary antibodies used were antibodies against β-actin, Cyclin D1 (DCS6), phospho-Cdc2 (Tyr15), Cenp-a (Cell Signaling Technologies, Beverly, MA) and Aurora-B kinase (Abcam, Cambridge, UK). Secondary antibodies used were alkaline phosphatase-conjugated mouse or rabbit anti-IgG (Promega, Madison, WI). Membranes were developed using colorimetric NBT-BCIP substrate (Promega), as described87 (link).
3
Comprehensive Cardiac Cell Identification Protocol
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The cells were fixed with 4% paraformaldehyde and permeabilized by 0.3% Triton X‐100 in blocking buffer with 5% goat serum in PBS for 1 h. Cells were then stained with primary antibodies, anti‐α‐sarcomeric actinin (Sigma), anti‐cardiac troponin T (DSHB), anti‐cardiac troponin I (DSHB), and anti‐α‐MHC (Abcam) for CM staining; anti‐vimentin (Santa‐Cruz) for CF staining. Ki‐67 (Genetex), histone H3 phosphorylated at serine 10 (Millipore), Aurora B kinase (Abcam) for gene screening. Secondary antibodies conjugated with Alexa fluor‐488 or Alexa fluor‐568 (Life Technology) were incubated for 1 h at room temperature after washing with PBS three times, and the nuclei were stained with DAPI (Life Technologies) for 5 min.
The tissue sections were deparaffinized, rehydrated, and antigens retrieved by boiling twice in sodium citrate solution. Then, the sections underwent the same immunofluorescence procedure as mentioned previously.
The tissue sections were deparaffinized, rehydrated, and antigens retrieved by boiling twice in sodium citrate solution. Then, the sections underwent the same immunofluorescence procedure as mentioned previously.
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