Anti cleaved caspase 3

Manufactured by Cell Signaling Technology

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Anti-cleaved caspase-3 is a lab equipment product that detects the cleaved form of caspase-3, a key executioner caspase involved in apoptosis.

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Lab products found in correlation

1 212 protocols using anti cleaved caspase 3

Multiparameter Analysis of Immune Cells in Murine Models

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For flow cytometry, we used anti-CD45, anti-CD11b, anti-MHCII, anti-CD11c, anti-F4/80, anti-TNFR1, anti-B220, anti-CD3, anti-CD4, anti-CD8 and anti-NK1.1 antibodies (all from BioLegend). For Western blotting, anti-MLKL (1:1000), anti-cleaved caspase 3 (1:1000), anti-cleaved caspase 8 (1:1000), anti-TNFRI (1:1000), β-actin (1:1000) and HRP conjugated secondary antibody (1:5000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-pMLKL (1:1000) was obtained from Sigma Aldrich (St. Louis, MO, USA). For confocal microscopy, we used anti-F4/80 (Abcam, Cambridge, UK), Anti-pMLKL (Sigma Aldrich), anti-cleaved caspase 3 and anti-TNFR1 (Cell Signaling Technologies). Secondary antibodies (goat antirat IgG [H+L], Alexa 647 [catalog A21247]; goat antirabbit IgG [H+L], Alexa Fluor 488 [catalog A11008]; and goat anti-mouse IgG [H+L], Alexa Fluor 594 [catalog A11032]) were obtained from Invitrogen (Waltham, MA, USA), and Fluoroshield mounting medium with DAPI (Abcam, catalog ab104139) was used to stain nuclei. For neutralization studies, an anti-TNFR1 antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Streptozotocin (STZ), Nicotinamide (NA), deoxyadenosine monophosphate, acetyl choline, zVAD-FMK, Nec-1 and shikonin were obtained from Millipore Sigma (St. Louis, MO, USA). Pyridoxine and 2-Ketohexanoic acid were purchased from Cayman Chemicals.

Vankayalapati A., Durojaye O., Mukherjee T., Paidipally P., Owusu-Afriyie B., Vankayalapati R, & Radhakrishnan R.K. (2024). Metabolic changes enhance necroptosis of type 2 diabetes mellitus mice infected with Mycobacterium tuberculosis. PLOS Pathogens, 20(5), e1012148.

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Immunohistochemical Analysis of Apoptosis and Mitosis

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For immunohistochemical analysis, anti-cleaved caspase 3 (monoclonal, Cell Signaling Technology, Inc., Danvers, MA, USA) and phospho-histone H3 (polyclonal, Cell Signaling Technology, Inc., Danvers, MA, USA) were used as primary antibodies. Immunohistochemical analysis was performed using a universal second antibody kit that uses a peroxidase-conjugated labelled dextran polymer (Envision Plus, Dako, Glostrup, Denmark). The following primary antibodies were used: anti-cleaved caspase 3 (monoclonal, Cell Signaling Technology, Inc., Danvers, MA, USA) and phospho-histone H3 (polyclonal, Cell Signaling Technology, Inc., Danvers, MA, USA).

Iglesias P., Seoane M., Golán-Cancela I., Fraga M., Arce V.M, & Costoya J.A. (2023). A New Opportunity for “Old” Molecules: Targeting PARP1 Activity through a Non-Enzymatic Mechanism. International Journal of Molecular Sciences, 24(10), 8849.

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Fetal Erythrocytes Immunohistochemistry

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Analysis of foetal erythrocytes from cord blood samples was carried out by Wright-Giemsa staining. For immunohistochemical analysis, anti-cleaved caspase 3 (monoclonal, Cell Signaling) and phospho-histone H3 (polyclonal, Cell Signaling Technology, Inc) were used as primary antibodies. Immunohistochemical analysis was performed using a universal second antibody kit that uses a peroxidase-conjugated labelled dextran polymer (Envision Plus, Dako, Glostrup, Denmark). The following primary antibodies were used: anti-cleaved caspase 3 (monoclonal, Cell Signaling Technology, Inc) and phospho-histone H3 (polyclonal, Cell Signaling Technology, Inc).

Iglesias P., Seoane M., Golán I., Castro-Piedras I., Fraga M., Arce V.M, & Costoya J.A. (2020). PARP1 Deficiency Reduces Tumour Growth by Decreasing E2F1 Hyperactivation: A Novel Mechanism in the Treatment of Cancer. Cancers, 12(10), 2907.

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Immunohistochemical Profiling of PDX Specimens

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Paraffin-embedding, sectioning, hematoxylin and eosin (H&E) and Masson’s trichrome stainings as well as immunohistochemical staining of PDX specimens were performed by the Robert H. Lurie Comprehensive Cancer Center Pathology Core Facility. Tumor sections were stained with anti-Cytokeratin 19 ((CK-19), #ab76539; Abcam), anti-Ki-67 (#GA626; Dako), anti-cleaved caspase 3 ((CC3, #9661; Cell Signaling Technology), anti-pan-RAS ((RAS), #PA5–85947; Thermo Fisher Scientific) and anti-Phospho-p44/42 MAPK ((ERK1/2) (Thr202/Tyr204, (D13.14.4E) XP, #4370; Cell Signaling Technology) antibodies as described previously ²⁰. Primary antibodies were detected using the appropriate secondary antibodies and 3,3′-diaminobenzidine (DAB) revelation (Dako).
All slides were scanned using a Nanozoomer HT slide scanner (Hamamatsu). Quantification of positively immunostained cells was performed using customized Application Protocol Packages (APPs) within the VIS image analysis software (Visiopharm). Positive cells were counted in the whole tumor sections and expressed as the percentage ratio over the area of the whole section. Because the pattern of cytoplasmic RAS staining was diffuse, we quantified RAS signal intensity by color deconvolution using ImageJ (Fiji version) as previously described ²⁹.

Vidimar V., Park M., Stubbs C.K., Ingram N.K., Qiang W., Zhang S., Gursel D., Melnyk R.A, & Satchell K.J. (2022). Proteolytic pan-RAS cleavage leads to tumor regression in patient-derived pancreatic cancer xenografts. Molecular cancer therapeutics, 21(5), 810-820.

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Immunohistochemical Analysis of Apoptosis

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Animals were anesthetized with ketamine/xylazine and perfused transcardially with saline followed by 4% paraformaldehyde. The brains were removed and postfixed in 4% paraformaldehyde for 2 h and cyroprotected in 30% sucrose overnight. Sections (12 µm) were cut on a cryostat (Leica) and mounted onto charged slides. Sections were blocked in PBS/5% BSA and permeabilized with PBS/0.3% Triton X-100, and then exposed to primary antibodies overnight at 4°C in PBS/1% BSA. Slides were then washed three times in PBS, exposed to secondary antibodies coupled to different fluorophores at room temperature for 1 h in the dark. Sections were washed again three times, with 4’,6’-diamidino-2-phenylindole (DAPI; Sigma; 1:10,000) present in the final wash. Sections were coverslipped with antifading medium (ProLong Gold; Invitrogen) and analyzed by fluorescence microscopy (Nikon). Primary antibodies used are as follows: anti-p75 (1:500; R&D Systems, RRID:AB_2298561) and anti-cleaved caspase-3 (CC3; 1:1000; Cell Signaling Technology, RRID:AB_2069869).

Greenwood S.G., Montroull L., Volosin M., Scharfman H.E., Teng K.K., Light M., Torkin R., Maxfield F., Hempstead B.L, & Friedman W.J. (2018). A Novel Neuroprotective Mechanism for Lithium That Prevents Association of the p75NTR-Sortilin Receptor Complex and Attenuates proNGF-Induced Neuronal Death In Vitro and In Vivo. eNeuro, 5(1), ENEURO.0257-17.2017.

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Quantitative Analysis of Phospho-PAK1/2/3 in MPNST

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Tisues were fixed overnight in 4% paraformaldehyde, dehydrated and embedded in paraffin according to standard protocols. IHC was performed with antibodies for anti-cleaved caspase-3 (CC3) and p-Histone H3 (S10) (pHH3) (Cell Signaling Technology).
A tissue microarray (TMA), containing specimens retrieved from human MPNSTs (n=207), neurofibromas (n=56) and normal nerves (n=11) surgical resections, was used to assess phospho-PAK1/2/3 levels. Tissue sections were blocked for 20 minutes and heat-induced antigen retrieval was performed in pH 6 Citrate Buffer for 20 minutes. Endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol for 10 minutes. The sections were incubated overnight with phopho-PAK1/2/3 primary antibodies (Abcam) at 4°C. Anti-rabbit secondary antibodies (Dako) were incubated for 1 h at room temperature and visualized using DAB+ chromogen (Dako). Images were captured using the Vectra Automated Quantitative Pathology Imaging System and analyzed using inForm image analysis software (PerkinElmer, Caliper Life Sciences).
All human subject studies were approved by respective institutional review boards.

Semenova G., Stepanova D.S., Dubyk C., Handorf E., Deyev S.M., Lazar A.J, & Chernoff J. (2017). Targeting Group I p21-Activated Kinases to Control Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis. Oncogene, 36(38), 5421-5431.

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Multidimensional Histological Analysis of Tumors

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Samples were stained with both standard hematoxylin and eosin (Merck, Darmstadt, Germany) (HE) and Masson trichrome (MT) (Sigma-Aldrich, St. Louis, MO) dyes. The HE-stained sections were used to quantify vital, necrotic, and fibrotic tissue fractions. The necrotic and fibrotic fractions were calculated as a percentage of the overall tissue area across each section. For this purpose, at least 10 different fields were investigated at a 400 × magnification.
Immunohistochemical analysis of tumors was performed using anti-γH2AX [rabbit polyclonal; Cell Signaling Technology (Beverly, MA), No. 2577, 1:50 in 1% BSA diluted in phosphate saline buffer], anti–cleaved caspase 3 (CC3) [rabbit polyclonal; Cell Signaling Technology, No. 9661, 1:100 in 1% phosphate-buffered saline with bovine serum albumin], and anti–Ki-67 probes (Dako (Glostrup, Denmark); 1:100). For evaluation of the amount of lipids, frozen sections were mounted on glass slides and stained with Oil Red O (Sigma-Aldrich, St. Louis, MO). All histopathology was evaluated by an experienced pathologist in a blinded study setting. The pathology findings were used to cross-validate the longitudinal changes in the optical end-points.

Spliethoff J.W., Evers D.J., Jaspers J.E., Hendriks B.H., Rottenberg S, & Ruers T.J. (2014). Monitoring of Tumor Response to Cisplatin Using Optical Spectroscopy. Translational Oncology, 7(2), 230-239.

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Mouse Brain Preparation and Immunohistochemistry

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Brains harvested from mice were post-fixed overnight in 4% PFA, cryoprotected in 30% sucrose, processed for OCT embedding and sectioned at 40 μm as previously described [33 (link)]. For immunohistochemistry, standard methods with antigen retrieval in sodium citrate were used [34 (link)]. Antibodies used in this study included: anti-Olig2 (Millipore 1:500), anti-myelin basic protein (MBP; Millipore 1:500), anti-Cleaved Caspase 3 (CC3; Cell Signaling 1:500), and anti-Nkx2.2 (Iowa Hybridoma 1:50). Immunoreactivity was detected by incubation with appropriate Alexa-conjugated secondary antibodies (Molecular Probes—Life Technologies). Images of brain sections were captured using a Nikon NIE fluorescent microscope. The number of immune-positive cells per 100 μm² area was quantified using Nikon Elements analysis software. Statistical significance was determined using Student’s t-test and presented as mean cells per field. All studies were blinded and performed on coded sections (n = 6 hypoxic + 6 normoxic animals for IHC and Western blotting (each)).

Watzlawik J.O., Kahoud R.J., O’Toole R.J., White K.A., Ogden A.R., Painter M.M., Wootla B., Papke L.M., Denic A., Weimer J.M., Carey W.A, & Rodriguez M. (2015). Abbreviated Exposure to Hypoxia Is Sufficient to Induce CNS Dysmyelination, Modulate Spinal Motor Neuron Composition, and Impair Motor Development in Neonatal Mice. PLoS ONE, 10(5), e0128007.

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Quantitative Analysis of Phospho-PAK1/2/3 in MPNST

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Immunohistochemical Analysis of Embryonic Brain

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Immunohistochemistry was carried out as described previously (Andrews et al. 2008 (link)). Dissected embryonic brains were fixed in 4% PFA, cryoprotected overnight in 30% sucrose and frozen embedded in OCT (Tissue Tek). Brains were sectioned on a Cryostat (Bright Instruments) at 25 μm and incubated overnight in one of the following antibodies: rabbit polyclonal anti-calbindin (CB-28; 1:3000; Swant), mouse monoclonal anti-Cd140b/Pdgfrß (1:350, ThermoFisher Scientific), chicken polyclonal raised against GFP (1:500, Aves Labs), rabbit anti-phosphohistone H-3 (PH3; 1:1000, Milipore), anti-cleaved caspase-3 (CC3; 1:250, Cell Signaling Technology), anti-VEGFR1, anti-VEGFR2, anti-VEGFR3 (all 1:500, R&D Systems), rabbit anti-human VEGF (1:350, Abcam), anti-rab Tbr2, anti-mouse Satb2, anti-rat Satb2 (all 1:350, Abcam), and anti-mG10 (1:3000; kind gift from A. Goffinet). For blood vessel staining, sections were incubated with biotinylated Griffonia (Bandeiraea) Simplicifolia lectin I (Isolectin B4) (1:200, Vector) followed by fluorescent Strepatividin-405 (1:200, Vector Lab).

Barber M., Andrews W.D., Memi F., Gardener P., Ciantar D., Tata M., Ruhrberg C, & Parnavelas J.G. (2018). Vascular-Derived Vegfa Promotes Cortical Interneuron Migration and Proximity to the Vasculature in the Developing Forebrain. Cerebral Cortex (New York, NY), 28(7), 2577-2593.

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