The treatment with pevonedistat was performed in a cell culture medium for 24 h. Following fixation with methanol, cells were permeabilized with 0.25% TritonX-100 and subjected to staining with anti-pH3 antibody (Cell Signaling, Danvers, MA, USA; diluted 1:50 in 5% bovine serum albumin (BSA)/0.2% Triton/phosphate-buffered saline (PBS)). Secondary antibody was goat anti-rabbit Alexa488-conjugated antibody (Invitrogen, A11034, Life Technology Europe BV, Zug, Switzerland), followed by co-staining with propidium iodide (PI). Data were acquired by a flow cytometer (Attune, Applied Biosystems, Life Technology Europe BV, Zug, Switzerland) and analyzed by FlowJo software v.10 (BD Biosciences, San Jose, CA, USA).
Anti ph3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-pH3 antibody is a laboratory reagent used to detect and quantify phosphorylated histone H3, a marker of cellular mitotic activity. This antibody specifically binds to the phosphorylated serine 10 residue of histone H3, allowing for the identification and analysis of cells undergoing mitosis.
Automatically generated - may contain errors
Lab products found in correlation
Anti actin antibody, by MP Biomedicals (2 mentions)
Alexa fluor 405 and 488 conjugated streptavidin, by Thermo Fisher Scientific (2 mentions)
Gsl 1 isolectin b4, by Vector Laboratories (2 mentions)
Irdye 700, by LI COR (1 mentions)
Anti plcβ1, by Santa Cruz Biotechnology (1 mentions)
Anti plc β2, by Santa Cruz Biotechnology (1 mentions)
Anti plc β3, by Santa Cruz Biotechnology (1 mentions)
Anti plc β4, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Cytiva (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Penicillin streptomycin, by Cytiva (1 mentions)
Trypsin edta, by Cytiva (1 mentions)
C10337, by Thermo Fisher Scientific (1 mentions)
Anti ki67 antibody, by Cell Signaling Technology (1 mentions)
Anti pparγ antibody, by Cell Signaling Technology (1 mentions)
Cy3 conjugated goat antirabbit secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Anti enos antibody, by BD (1 mentions)
Blocking buffer, by Cell Signaling Technology (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti ph3 antibody
1
Pevonedistat Treatment and Cell Cycle Analysis
2
Endothelial Cell Culture Reagents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All endothelial cell culture media were obtained from Lonza, Inc. (Walkersville, MD, USA). Dulbecco's modified Eagle's medium, fetal bovine serum, trypsin-EDTA and penicillin/streptomycin (antibiotics) were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Anti-PLC-β1, anti-PLC-β2, anti-PLC-β3 and anti-PLC-β4 were obtained from Santa Cruz (California City, CA, USA). Anti-actin antibody was purchased from MP Biomedicals (Aurora, OH, USA). NG2 antibody was obtained from Millipore Bioscience (Temecula, CA, USA). Anti-pH3 antibody was purchased from Cell Signaling Technology (Boston, MA, USA). GSL I-isolectin B4 was obtained from Vector Laboratories (Burlingame, CA, USA). Alexa Fluor 405- and 488-conjugated streptavidin, and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies were purchased from Molecular Probes, Inc. (Carlsbad, CA, USA). IRDye700- and IRDye800-conjugated rabbit and mouse secondary antibodies were obtained from Li-COR Bioscience (Lincoln, NE, USA).
3
Quantifying Cell Cycle Dynamics in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For EdU pulse-chase experiments, worms were fed E. coli MG1693 that had been grown in minimal medium supplemented with glucose [81 (link)] and 75 mM of the thymidine analog EdU (C10337, Life Technologies, Grand Island, NY). Immediately following seeding, plates were stored at 4 °C. Plates were warmed to 20 °C prior to use. Worms were kept for either 15 or 30 minutes on EdU-labeled bacteria in the dark, returned to non-labeled bacteria in the dark for the period of the chase, and were fixed and processed as described [26 (link)] using 0.1 μg/ml DAPI to label DNA and 1:200 anti-PH3 antibody (9706, Cell Signaling, Beverly, MA) followed by Alexa 594-conjugated anti-mouse antibody (A21203, Life Technologies, Grand Island, NY) to label M-phase cells.
CYE-1 and PGL-1 stainings were performed by freeze-cracking dissected gonads or whole larvae, dehydration in acetone, 5-minute fixation in 4 % PFA, incubation with anti-CYE-1 antibody (a gift from Edward Kipreos) at 1:5 dilution or rabbit anti-PGL-1 antibody (a gift from Susan Strome) at a 1:500 dilution, and incubation with DAPI and Alexa 594-conjugated anti-mouse or anti-rabbit secondary antibodies.
All samples were imaged at ~0.3-μm z intervals with LSM 710 or 780 confocal microscopes (Carl Zeiss MicroImaging, Oberkochen, Germany), using a 63× objective.
CYE-1 and PGL-1 stainings were performed by freeze-cracking dissected gonads or whole larvae, dehydration in acetone, 5-minute fixation in 4 % PFA, incubation with anti-CYE-1 antibody (a gift from Edward Kipreos) at 1:5 dilution or rabbit anti-PGL-1 antibody (a gift from Susan Strome) at a 1:500 dilution, and incubation with DAPI and Alexa 594-conjugated anti-mouse or anti-rabbit secondary antibodies.
All samples were imaged at ~0.3-μm z intervals with LSM 710 or 780 confocal microscopes (Carl Zeiss MicroImaging, Oberkochen, Germany), using a 63× objective.
4
Quantifying Hepatocyte Proliferation in Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissue was fixed overnight in 4% PFA, embedded in paraffin, and sectioned in 3 μm slices. immunohistochemistry (IHC) was performed with the following antibodies: anti-Ki67 antibody (1:500, Cell Signaling Technology), anti-PH3 antibody (1:400, Cell Signaling Technology), and anti-PPARγ antibody (1:1000, Cell Signaling Technology). They all were counterstained with hematoxylin and eosin (H&E). Hepatocyte proliferation was determined by quantification of Ki67- and PH3-positive cells. The percentage of proliferative hepatocyte was determined by examination of at least four random 100x fields in three different sections. At least 200 nuclei were counted in each field. For morphology assessment, the sections were also stained with H&E.
5
Immunostaining Antibodies and Reagents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-eNOS antibody was purchased from BD Biosciences (San Jose, CA, USA). Anti-SM22a antibody was obtained from Abcam (Cambridge, UK). Anti-actin antibody was purchased from MP Biomedicals (Aurora, OH, USA). NG2 antibody was obtained from Millipore Bioscience (Temecula, CA, USA). Anti-pH3 antibody was purchased from Cell Signaling Technology (Boston, MA, USA). GSL I-isolectin B4 was obtained from Vector Laboratories (Burlingame, CA, USA). Alexa Fluor 405- and 488-conjugated streptavidin, and Cy3-conjugated goat anti-rabbit secondary antibodies were purchased from Molecular Probes, Inc. (Carlsbad, CA, USA). IRDye700- and IRDye800-conjugated rabbit and mouse secondary antibodies were obtained from Li-COR Bioscience (Lincoln, NE, USA).
6
Zebrafish Embryo Cell Cycle Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zebrafish embryos were dissected into two parts, the anterior parts were used for genotyping and the posterior parts were fixed in 4% PFA at 4°C overnight and then dehydrated with methanol at -20°C for 30 min. After washing with PBST buffer (PBS with 0.1% Tween 20) four times (5 min each), the embryos were digested within 50 μM proteinase K for 1 h. The permeabilized embryos were re-fixed with 4% PFA for 20 min. After washing with PBST buffer three times on a shaker, the embryos were blocked with 1% blocking buffer (Roche) for 2 h at room temperature and then incubated with anti-pH3 antibody diluted in blocking buffer (1:500, Cell Signaling) overnight. On the next day, the embryos were washed three times (15 min) with PBST buffer and incubated with a secondary fluorescent antibody (Invitrogen) overnight. Finally, the embryos were washed three times (15 min) with PBST buffer and photographed by confocal microscopy (Nikon, A1R + ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!