Rabbit anti cleaved caspase 3 antibody

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

The Rabbit anti-cleaved caspase-3 antibody is a primary antibody that recognizes the cleaved form of caspase-3 protein. Caspase-3 is a key executioner enzyme in the apoptosis (programmed cell death) pathway.

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Lab products found in correlation

Rabbit anti caspase 3 antibody, by Cell Signaling Technology (4 mentions) Oct compound, by Sakura Finetek (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Staurosporine, by Thermo Fisher Scientific (2 mentions) Caspase 8 inhibitor 2, by Merck Group (2 mentions) Z fa fmk, by BD (2 mentions) Rabbit anti cleaved caspase 8 antibody, by Cell Signaling Technology (2 mentions) Rabbit anti cleaved caspase 9 antibody, by Cell Signaling Technology (2 mentions) Anti mouse thy 1, by Bio-Rad (2 mentions) Anti α actin polyclonal antibody, by Santa Cruz Biotechnology (2 mentions) Rabbit anti parp antibody, by Cell Signaling Technology (2 mentions) Apo direct kit, by BD (2 mentions) Protein g agarose, by Roche (2 mentions) Biotinylated rabbit secondary antibody, by Vector Laboratories (2 mentions) Rabbit anti cd31, by Cell Signaling Technology (2 mentions) Antigen unmasking solution h 3300, by Vector Laboratories (2 mentions) Immpact dab peroxidase hrp substrate, by Vector Laboratories (2 mentions) Nanozoomer 2.0 ht, by Hamamatsu Photonics (2 mentions) Alexa fluor 568, by Thermo Fisher Scientific (2 mentions) Lsm 880 confocal microscope, by Zeiss (2 mentions)

Close spelling variants of this product

Rabbit anti-cleaved caspase-3 primary antibody Rabbit polyclonal anti-cleaved caspase 3 (Asp175) antibody Rabbit anti-human cleaved caspase 3 (Asp175) polyclonal antibody Rabbit monoclonal anti-cleaved caspase-3 antibody Rabbit anti-mouse cleaved caspase-3 antibody Rabbit anti-cleaved caspase-3 (Asp175) antibody Rabbit anti-cleaved caspase 3 polyclonal antibody Alexa-Fluor-647-conjugated rabbit anti-cleaved caspase-3 (9602S) antibody Rabbit anti-human cleaved caspase-3 antibody Rabbit anti-mouse cleaved caspase-3 (Asp175) antibody

49 protocols using rabbit anti cleaved caspase 3 antibody

Immunofluorescence Analysis of Vascular Endothelial Cells and Apoptosis

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Fixed specimens were embedded in OCT compound (Sakura Finetek, Tokyo, Japan) and sectioned (10-μm thick slices). For immunofluorescence analyses, rat anti-CD31 antibody (1/200) (BD Biosciences, San Diego, CA, USA) was used to stain vascular endothelial cells and rabbit anti-cleaved caspase-3 antibody (1/200) (#9579, Cell Signaling Technology, Danvers, MA, USA) to stain apoptotic cells. Anti-rat IgG Alexa Fluor 488 and anti-rabbit IgG Alexa Fluor 546 (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. Samples were visualized using a Leica DM5500B and processed with the Leica application suite (Leica Microsystems, Bensheim, Germany). All images shown are representative of more than 3 independent experiments. Vessels and apoptotic cells were counted in more than 3 different fields in each tumor at a magnification of ×100.

Kanzaki R., Naito H., Kise K., Takara K., Eino D., Minami M., Shintani Y., Funaki S., Kawamura T., Kimura T., Okumura M, & Takakura N. (2017). Gas6 derived from cancer-associated fibroblasts promotes migration of Axl-expressing lung cancer cells during chemotherapy. Scientific Reports, 7, 10613.

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Inhibition of JNK Signaling in Immune Cells

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We purchased JNK inhibitor SP600125 from Cayman Chemical (Ann Arbor, MI, USA), and transforming growth factor alpha (TGF‐α) from R&D Systems (Minneapolis, MN, USA). Primary antibodies used in immunohistochemical and immunofluorescence assays included rabbit anti‐phospho‐JNK antibody (1:50; Cell Signaling Technology, Danvers, MA, USA), rat anti‐mouse CD45 antibody (1:50; BD Biosciences, San Jose, CA, USA), rat anti‐mouse F4/80 antibody (1:100; eBioscience, San Diego, CA, USA), rabbit anti‐Ki‐67 antibody (1:100; Abcam, Cambridge, UK), mouse anti‐α‐smooth muscle actin (α‐SMA) antibody (1:50; Santa Cruz, Santa Cruz, CA, USA), biotin hamster anti‐mouse CD11c antibody (1:100; BD Biosciences), rabbit anti‐cleaved caspase3 antibody (1:1600; Cell Signaling Technology) and rabbit anti‐CD8 antibody (clone EP1150Y, 1:250; Epitomics, Burlingame, CA, USA). Primary antibodies used in immunoblotting analysis included rabbit anti‐phospho‐JNK antibody (1:1000; Cell Signaling Technology), rabbit anti‐JNK antibody (1:1000; Cell Signaling Technology), rabbit anti‐phospho‐Stat3 antibody (1:2000; Cell Signaling Technology) and mouse anti‐Stat3 antibody (1:1000; Cell Signaling Technology).

Sato T., Shibata W., Hikiba Y., Kaneta Y., Suzuki N., Ihara S., Ishii Y., Sue S., Kameta E., Sugimori M., Yamada H., Kaneko H., Sasaki T., Ishii T., Tamura T., Kondo M, & Maeda S. (2017). c‐Jun N‐terminal kinase in pancreatic tumor stroma augments tumor development in mice. Cancer Science, 108(11), 2156-2165.

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Apoptosis Induction and Signaling Pathway

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Soluble human recombinant FasL (rhFasL) (Kamiya Biomedical Company, Seattle, WA); staurosporine (ACROS Organics, Pittsburgh, PA); caspase 8 inhibitor II (EMD Millipore, Billerica, MA); protein G-agarose (Roche Diagnostics, Indianapolis, IN); Z-FA-FMK (negative control for caspase inhibitors), FITC Rat anti-mouse CD90.2, FITC Rat IgG1, κ Isotype, Annexin V apoptosis detection kit and APO-Direct kit (BD Pharmingen, San Jose, CA); rabbit anti-PARP antibody, rabbit anti-caspase 3 antibody, rabbit anti-cleaved caspase 3 antibody, rabbit anti-cleaved caspase 8 antibody and rabbit anti-cleaved caspase 9 antibody (Cell Signaling Technologies, Danvers, MA); anti-mouse Thy-1 (AbD Serotec, Oxfordshire, UK), and anti-α actin polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX).

Liu X., Wong S.S., Taype C.A., Kim J., Shentu T.P., Espinoza C.R., Finley C., Bradley J.E., Head B.P., Patel H.H., Mah E.J, & Hagood J.S. (2017). Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution. Laboratory investigation; a journal of technical methods and pathology, 97(3), 256-267.

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Immunohistochemical Detection of Cleaved Caspase-3

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Paraffined sections were rehydrated and washed in 0.01-M phosphate buffered saline (PBS). Then, they were incubated over night at 4°C with a rabbit anti-cleaved caspase-3 antibody (Cell Signalling Technology, Inc., Danvers, USA) at 1∶300 dilution. Then, ocular sections were washed in PBS and incubated with the specific secondary antibody biotinylated anti-rabbit IgG (1∶100) (Vector Laboratories, Burlingame, USA). Once washed in PBS, a streptavidin Alexa Fluor 488 conjugate (Molecular Probes-Life Technologies, Grand Island, USA) at 1∶100 dilution was used to detect cleaved caspase-3 immunolabelling; the incubation was made over night at 4°C. Nuclear counterstaining with Hoescht stain solution (Sigma-Aldrich Chemie, Buchs, Switzerland) was performed for microscopic analysis with the laser scanning confocal microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany). Negative control was carried out by omitting the primary antibody. Specific labeling of cleaved caspase-3 antibody in mouse jejunal paraffin sections was used as positive control.

Bogdanov P., Corraliza L., A. Villena J., Carvalho A.R., Garcia-Arumí J., Ramos D., Ruberte J., Simó R, & Hernández C. (2014). The db/db Mouse: A Useful Model for the Study of Diabetic Retinal Neurodegeneration. PLoS ONE, 9(5), e97302.

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Mammosphere Proliferation and Apoptosis Assay

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Immunofluorescent staining of Ki67 and cleaved caspase 3 (CC3) was performed to phenotype our mammospheres based on proliferation (Ki67 staining) and apoptosis (CC3 staining) levels. Mammospheres were centrifuged (5 min, 1500G) onto poly-D-lysine coated coverslips (Neuvitro H-12-1.5-PDL). Cells were fixed with 4% paraformaldehyde (ThermoFischer 28908) in PBS for 10 minutes, permeabilized with 0.05% Triton-X-100 (Sigma X-100) for 20 minutes, and then non-specific binding was blocked with 10% normal goat serum (Invitrogen PCN5000) for one hour. To detect Ki67, cells were incubated with an anti-Kit67 antibody conjugated to Alexa Fluor 488 (1:100 dilution; BD Pharmingen #561165). To detect cleaved caspase-3, cells were incubated with rabbit anti-Cleaved caspase 3 antibody (1:300; Cell Signaling Technologies 9661), followed by a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 568 (1:200; ThermoFischer #A-11011). Following incubation with antibodies, cells were mounted with ProLong® Gold Antifade Reagent with DAPI (Cell Signaling Technology 8961).

Madonna M.C., Fox D.B., Crouch B.T., Lee J., Zhu C., Martinez A.F., Alvarez J.V, & Ramanujam N. (2019). Optical imaging of glucose uptake and mitochondrial membrane potential to characterize Her2 breast tumor metabolic phenotypes. Molecular cancer research : MCR, 17(7), 1545-1555.

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Immunostaining of Tumor Caspase-3

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Animals were killed 25 days after inoculation, and their tumors were embedded in the OCT compound (Sakura Finetek, Torrance, CA, USA) and stored at −80 °C. Frozen sections (10 μm thick) were fixed in 4% paraformaldehyde/phosphate-buffered saline and subjected to immunostaining. Immunostaining was performed using a rabbit anticleaved caspase-3 antibody (Cell Signaling Technology), as described previously [39 (link)]. The nuclei were counterstained with Hoechst 33342, and the sections were observed by confocal microscopy (Carl Zeiss, Oberkochen, Germany).

Yoshino S., Hara T., Nakaoka H.J., Kanamori A., Murakami Y., Seiki M, & Sakamoto T. (2016). The ERK signaling target RNF126 regulates anoikis resistance in cancer cells by changing the mitochondrial metabolic flux. Cell Discovery, 2, 16019-.

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Immunohistochemistry for Cleaved Caspase-3

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Immunohistochemistry for cleaved caspase-3 was done as explained next [6 (link),17 (link)]. The sections were treated with rabbit anticleaved caspase-3 antibody (1:500; Cell Signaling Technology, Inc.) during overnight, and then incubated with biotinylated rabbit secondary antibody (1:200; Vector Laboratories) for another 1 hour. After amplification of the secondary antibody with the Vector Elite ABC kit (1:100; Vector Laboratories), 0.03% diaminobenzidine was used to visualized the antibodybiotin-avidin-peroxidase complex.

Ko Y.J, & Ko I.G. (2020). Voluntary Wheel Running Improves Spatial Learning Memory by Suppressing Inflammation and Apoptosis via Inactivation of Nuclear Factor Kappa B in Brain Inflammation Rats. International Neurourology Journal, 24(Suppl 2), 96-103.

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Apoptosis Induction and Quantification

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After 24 h and 48 h, uninfected and infected HeLa cells were collected after trypsinization and resuspended in REPA buffer (50 mM Tris, 150 mM Nacl, 0.1% SDS, 0.5% Sodium Deoxycholate, 1% Triton X100, 1 mM PMSF) containing 1x protease inhibitor cocktail (Sigma) to lyse the eukaryotic cells. The lysate was then loaded onto SDS-PAGE gels and transferred to a PVDF membrane (Millipore) through Western blotting. Blots were blocked using 5% non-fat milk (Carl Roth) and probed with rabbit anti-cleaved caspase 3 antibody (1:1000) (Cell Signalling) or rabbit anti-vinculin antibody (1:1000) (Santa Cruz Biotech). After incubating with horseradish peroxidase-conjugated rabbit IgG secondary antibody (Abcam), blots were processed using the enhanced chemiluminescence (ECL) Western blotting detection system (Millipore). Images were taken using Gel Doc™ XR+ System (Bio-Rad). Bands were quantified using ImageJ software (National Institutes of Health, USA) and normalised against loading control (vinculin). Mean optical density values were plotted using GraphPad Prism 5 (Graphpad Software).

Hegde S., Hegde S.M., Rosengarten R, & Chopra-Dewasthaly R. (2016). Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro. PLoS ONE, 11(9), e0163603.

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Western Blot Analysis of Post-Stroke Proteins

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Perihematomal tissues from the ipsilateral hemisphere were homogenized and centrifuged, then separated using SDS-poly-acrylamide gel electrophoresis, and transmitted to nitrocellulose membranes. Next, we blocked the membrane with defatted milk (5%) for 1 h and incubated it overnight at 4 °C with the following primary antibodies: rabbit anti-HDAC6 antibody (1:2,000, #ab239362; Abcam, Cambridge, UK), mouse anti-ace-α-tubulin (1:2,000, #ab24610; Abcam, Cambridge, UK), rabbit anti-cleaved caspase-3 antibody (1:1,000, #9661; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Bax antibody (1:1,000, #ab32503; Abcam, Cambridge, UK), rabbit anti-Bcl-2 antibody (1:1,000, #ab196495; Abcam, Cambridge, UK), or mouse anti-β-actin antibody (1:5,000, #66009-1-Ig; Proteintech, Chicago, IL, USA). Corresponding secondary antibodies were chosen to incubate for 90 min. The data were analyzed using Image J software.

Peng C., Gong X., Hu Z., Chen C, & Jiang Z. (2023). Selective HDAC6 inhibitor TubA offers neuroprotection after intracerebral hemorrhage via inhibiting neuronal apoptosis. PeerJ, 11, e15293.

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Western Blot Analysis of PARP and Caspase-3

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PolyADP-ribose polymerase (PARP) and caspase-3 cleavage were assessed using a standard western blotting protocol [17] (link). Membranes were probed using polyclonal rabbit anti-PARP1 antibody (Abcam, Cambridge, Cambridgeshire, UK; 1∶1000), a rabbit monoclonal anti-ERCC1 antibody (Cell Signaling Technology, Danvers, MA, USA; 1∶1000) and a polyclonal rabbit anti-cleaved caspase-3 antibody (Cell Signaling Technology; 1∶1000). A rabbit anti-actin antibody (Sigma-Aldrich Co. Ltd; 1∶2000) was used as a loading control. Blots were scanned (Bio-Rad GS-800 Calibrated Densitometer; Bio-Rad, Hercules, CA, USA) and signal quantification was performed by densitometry using scanning analysis software (Quantity One; Bio-Rad).

Witney T.H., Fortt R.R, & Aboagye E.O. (2014). Preclinical Assessment of Carboplatin Treatment Efficacy in Lung Cancer by 18F-ICMT-11-Positron Emission Tomography. PLoS ONE, 9(3), e91694.

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