Cell lysates were prepared and 20 µg of these were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Specific monoclonal anti-cleaved caspase-3 [Cell Signal Technology (CST), SN: 4380, dilution: 1:2000], monoclonal anti-Bcl-2 (CST, SN: 11988, dilution: 1:2000), monoclonal anti-p65 (CST, SN: 5741, dilution: 1:2000), monoclonal anti-p-p38 (CST, SN: 3195, dilution: 1:2000), and monoclonal anti-β-actin (CST, SN: 8457, dilution: 1:4000) antibodies were used. HRP-conjugated immunoglobulin was used as the secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). West Pico chemiluminescence was used as the substrate to visualize protein bands, which were quantified using densitometric image analysis software (Image Master VDS; Pharmacia Biotech) and normalized to β-actin expression.
Monoclonal anti cleaved caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
Monoclonal anti-cleaved caspase-3 is a laboratory antibody product. It specifically recognizes the cleaved form of caspase-3, a key enzyme involved in apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
Monoclonal anti β actin, by Cell Signaling Technology (1 mentions)
Monoclonal anti bcl 2, by Cell Signaling Technology (1 mentions)
Hrp conjugated immunoglobulin, by Jackson ImmunoResearch (1 mentions)
Image master vds, by GE Healthcare (1 mentions)
Monoclonal anti ki67, by Cell Signaling Technology (1 mentions)
Onestep polymer hrp conjugated anti mouse rat rabbit igg secondary antibody, by GeneTex (1 mentions)
Bx61 microscope, by Olympus (1 mentions)
Gfap antibody, by Cell Signaling Technology (1 mentions)
Monoclonal anti bax antibody, by Cell Signaling Technology (1 mentions)
Microamp fast optical 96 well reaction plate with barcode, by Thermo Fisher Scientific (1 mentions)
Antifade reagent, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Monoclonal anti p53, by Cell Signaling Technology (1 mentions)
4 protocols using monoclonal anti cleaved caspase 3
1
Western Blot Analysis of Apoptosis and Signaling
2
Immunohistochemical Analysis of Tumor Markers
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Fresh tumors tissue were fixed in 4% formaldehyde for 24 h, paraffin-embedded, and sectioned (at ~6 μM). Sections were treated with boiling ddH2O for antigen retrieving, 10 min. Endogenous peroxidase activity was inhibited with 3% H2O2 for 10 min at room temperature, and sections were then stained with antibodies against monoclonal anti-Ki67 (#12202; 1:400; Cell Signaling) and monoclonal anti-cleaved caspase-3 (#9664; 1:100; Cell Signaling), at 37°C for 3 h, followed by OneStep Polymer HRP-conjugated anti-mouse/rat/rabbit IgG secondary antibody (GTX83398; GeneTex) for 30 min at room temperature and then visualized using a colorimetric method (DAB kit; GTX30939; GeneTex). Nuclei were counterstained with hematoxylin and photographed using an Olympus BX61 microscope (Tokyo, Japan).
3
Immunohistochemistry and RT-PCR Assays
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Antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), including: monoclonal anti-glial fibrillary acidic protein (GFAP) antibody, monoclonal anti-cleaved caspase-3, monoclonal anti-bcl2 antibody, monoclonal anti-bax antibody, monoclonal anti-NeuN antibody and monoclonal anti-GAPDH antibody. Monoclonal anti-Tau from Millipore (Billerica, MA, USA), monoclonal anti-Tuj1 from Beyotime (Shanghai, China), monoclonal anti-Calbindin from SWANT (Bellinzona, Ticino, Switzerland). The RT-PCR and kits were purchased from TaKaRa (Shiga, Japan). Experimental materials including: MicroAmp® fast 8-tube strip and optical 8-cap strip, MicroAmp® fast optical 96 well reaction plate with barcode, MicroAmp® 96-well optical adhesive film were from Applied BiosystemsTM, Life Technologies Co. (Grand Island, NY, USA). Nucleic acid electrophoresis agarose and trizol were purchased from InvitrogenTM, Life Technologies Co.(Grand Island, NY, USA).
4
Malformin C Effects on p53, Caspase 3, and LC3
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Colon 38 cells were treated with different concentrations of Malformin C for three different time courses including 24-hour treatment, 48-hour treatment, and 24-hour treatment followed by Malformin C withdrawal and another 24-hour incubation. All the cells were fixed with 4% paraformaldehyde in PBS and then permeabilized with 0.5% Triton X-100 in PBS. 3% bovine serum albumin in PBS was used to block nonspecific binding. Cells were then exposed to monoclonal anti-p53, monoclonal anti-cleaved CASPASE 3, or monoclonal anti-LC3 (1:200; Cell Signaling Technology) at RT for 1 hour, washed with 0.2% Tween20 in PBS and followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG at 1:400 dilution. Cytoplasmic actin was counter-stained with 0.25μg/ml rhodamine phalloidin (Invitrogen). The cells were then sealed in antifade reagent (Invitrogen). Confocal micrographs were scanned by a laser scanning confocal microscope (LSM 510; Carl Zeiss, Inc., Thornwood, NY).
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