3x105 Tumor cells per well were seeded on 24mm square cover slips in 6-well plates and allowed to grow for 24 hours. Tumor cells were then treated with RCM1, VCR or RCM1 + VCR for 24 hours. Tumor cells were then fixed and stained as previously described (36 (link)). To visualize the nucleus, Hoechst 33342 (ThermoFisher Scientific) was used as a counter stain. For quantification, 5 random fields were acquired per sample and quantified using ImageJ. To perform immunostaining of tumor tissue, paraffin embedded Rd76-9 subcutaneous tumor sections were stained as described previously (37 (link)). 5 Random fields per sample were acquired and quantified using ImageJ. Antibodies used for immunostaining were anti-Ki-67 (Invitrogen, MA5-14520), anti-phospho-histone H3 (Santa Cruz, sc-374669 (C-2)), anti-CD31 (R&D, AF3628), anti-Cleaved-Caspase 3 (R&D, MAB835), anti-CHAC1 (Novus Biologicals, OTI1E2).
Anti phospho histone h3
Manufactured by Santa Cruz Biotechnology
Sourced in Germany, United States
Anti-phospho-histone H3 is a laboratory reagent used to detect phosphorylation of histone H3, which is a marker of cell division and chromatin condensation. It can be used in various techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to analyze the cell cycle and mitotic activity of samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti myogenin, by Santa Cruz Biotechnology (2 mentions)
Anti p21cip1, by Cell Signaling Technology (2 mentions)
Anti ki67, by Santa Cruz Biotechnology (2 mentions)
Anti cyclin d1, by BD (2 mentions)
Anti p27kip1, by Cell Signaling Technology (2 mentions)
Anti prb, by Cell Signaling Technology (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Af3628, by R&D Systems (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Ma5 14520, by Santa Cruz Biotechnology (1 mentions)
Anti cleaved caspase 3, by Merck Group (1 mentions)
Vibratome vt1000, by Leica (1 mentions)
Anti doublecortin, by Santa Cruz Biotechnology (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Anti mcherry, by Takara Bio (1 mentions)
7 protocols using anti phospho histone h3
1
Quantification of Tumor Cell Response
2
Neuroanatomical Assessment of Mouse Brain
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Thirty micrometers thick coronal sections were made using a VT1000 vibratome (Leica Biosystems). Immunofluorescence stainings were prepared using the following primary antibodies: forebrain: anti-CNPase (No. PA5-19551, Thermo Fisher Scientific, Waltham, MA, USA), cerebellum: anti-Pcp4 (No. sc-74816, Santa Cruz Biotechnology, Dallas, TX, USA). The thickness of different brain structures was measured on a series of six consecutive sections starting at Bregma −1.94 mm. To assess adult hippocampal neurogenesis the following antibodies were used: anti-phosphohistone H3 (No. sc-8656-R, Santa Cruz Biotechnology), anti-doublecortin (No. sc-8066, Santa Cruz Biotechnology), anti-cleaved caspase 3 (No. AB3623, Millipore, Merck, Darmstadt, Germany). To assess Purkinje cell density a region-of-interest of 600 × 600 µm was superimposed on the 6th cerebellar lobule (starting at ~ Bregma −6.6 mm) and the number of Purkinje cell profiles was determined. See supplementary information for a detailed description.
3
Evaluation of Cellular Responses Using Western Blot
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The Dulbecco's modified Eagle's medium (DMEM), MTT, hematoxylin and eosin were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). The fetal bovine serum (FBS) and antibiotics were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) and Immobilon P polyvinylidene fluoride membranes from Merck Millipore. OOS was provided by Catalysis, S.L. (Madrid, Spain). The other generic chemicals were purchased from Sigma-Aldrich, Roche Applied Science (Mannheim, Germany) or Merck Millipore.
The origins of the various antibodies used in the Western blot analyses are as follows: Anti-GAPDH (cat. no. sc-166574; 1:10,000) and anti-cyclin B (cat. no. sc-245; 1:5,000) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the anti-phospho-histone H3 (cat. no. 06-570; 1:3,500) from Merck Millipore. The horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit; cat. no. 170-6515; 1:20,000; and goat anti-mouse; cat. no. 170-6516; 1:10,000), were obtained from Bio-Rad Laboratories Inc. (Hercules, CA, USA).
The origins of the various antibodies used in the Western blot analyses are as follows: Anti-GAPDH (cat. no. sc-166574; 1:10,000) and anti-cyclin B (cat. no. sc-245; 1:5,000) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the anti-phospho-histone H3 (cat. no. 06-570; 1:3,500) from Merck Millipore. The horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit; cat. no. 170-6515; 1:20,000; and goat anti-mouse; cat. no. 170-6516; 1:10,000), were obtained from Bio-Rad Laboratories Inc. (Hercules, CA, USA).
4
Analyzing Anticancer Drug Effects on Cell Cycle
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PC3 cells were grown in RPMI medium, and 7.5 × 104 cells were added in 24-well culture plates containing slides and allowed to attach overnight at 37°C in 5% CO2. Then, they were treated with 6MPTOX (0.005 μM), PTOX (0.002 μM), and DMSO (2.25 μM) at 37°C for 72 h. The cells were fixed with PFA (p-formaldehyde) 4% in PEM buffer PIPES (0.1 M), EGTA (2 mM), and MgSO4 (1 mM), pH 6.95. After 15 min PFA/NaHCO3 was added and incubated for 45 min at room temperature. The slides were rinsed with PBS, treated with 0.1% Triton X-100 (Sigma Aldrich), and then incubated with the primary antibody antiphospho-histone H3 (1 : 500, Santa Cruz Biotechnology) overnight at 4°C. A secondary antibody anti-rabbit Alexa 488 (1 : 1000, Molecular Probes) was added and incubated for 1 h at 37°C. The cells were stained with 0.4 μg/mL 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR) in PBS for 10 min, then mounted, and imaged by fluorescence microscopy.
5
Immunohistochemical Analysis of Muscle Tissue
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Paraffin sections (5μm) were cut and stained with hematoxylin and eosin (H&E) for morphological examination. Immunostaining was performed using following antibodies: anti-myogenin, anti-Ki67, anti-phospho-Histone H3 (Santa Cruz), anti-pRb, anti-p27Kip1, anti-p21Cip1 (Cell Signaling Technology, Inc.), anti-Cyclin D1 (BD biosciences) were done as previously described (52 (link), 53 (link)).
6
Whole-Mount Immunohistochemistry of Fish Fins
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The fins were fixed in 4% paraformaldehyde overnight at 4°C and used for whole-mount immunohistochemistry with antiphospho-histone H3 (No. SC-8656-R; Santa Cruz Biotechnology, Inc., Dallas, TX) to detect proliferative cells, antiacetylated tubulin (No. T7451; Sigma-Aldrich, Saint Louis, MO) to detect axons, anti-GFP to detect GFP in Shh:GFP fish (No. ab13970; Abcam, Cambridge, MA), anti-mCherry (No. 6332543; Clontech Laboratories, Inc., Mountain View, CA) to detect mRFP in sox10:RFP fish, and anti-Sox10 (No. GTX128374; GeneTex, Inc., Irvine, CA). The P-H3-positive cells were counted in ray and inter-ray two in all segments. Immunofluorescence images were acquired using an inverted Leica SP5 with a Leica PL APO 20×/N.A. = 0.7 oil immersion objective. For coimmunolabeling of Sox10 and HH-positive cells, the fins were snap-frozen in optimal cutting temperature compound and sectioned at 20 μM with a cryomicrotome (No. HM560; Thermo Fisher Scientific, Waltham, MA).
7
Immunohistochemical Analysis of Muscle Tissue
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