Rabbit anti cleaved caspase 3

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, Germany, China, Japan

Rabbit anti-cleaved caspase-3 is an antibody that specifically recognizes the cleaved form of caspase-3, a critical executioner caspase involved in the apoptosis (programmed cell death) pathway. This antibody can be used to detect and quantify the level of cleaved caspase-3 in various cell and tissue samples.

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360 protocols using rabbit anti cleaved caspase 3

Melanoma Spheres: BrdU, Apoptosis, and SOX2

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BrdU pulses in melanoma spheres were performed for 8 h (4 μg/ml). Spheres were plated on DMEM/Matrigel (1:40) (BD Biosciences) to allow attachment, fixed with 4% paraformaldehyde and processed as previously described.^{60 (link)} For apoptosis, spheres were immunolabeled with a rabbit anti-cleaved Caspase3 (Cell Signaling Technologies, Danvers, MA, USA), followed by an anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody. For SOX2 immunofluorescence, spheres were immunolabeled with a rabbit anti-SOX2 antibody (AB5603; Millipore, Billerica, MA, USA) and with an anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody. Formalin-fixed paraffin-embedded sections were subjected to antigen retrieval (with citrate buffer pH 6.0), incubated with rabbit anti-SOX2 antibody (AB5603; Millipore) and rabbit anti-cleaved Caspase3 (Cell Signaling Technologies) and visualized using UltraVision Detection System (Lab Vision, Fremont, CA, USA) and diaminobenzidine (Dako, Carpinteria, CA, USA).

Santini R., Pietrobono S., Pandolfi S., Montagnani V., D'Amico M., Penachioni J.Y., Vinci M.C., Borgognoni L, & Stecca B. (2014). SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells. Oncogene, 33(38), 4697-4708.

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Comprehensive Immunohistochemistry and Western Blot Antibody Panel

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The following antibodies were used in IHC and IF: guinea pig anti-insulin (DAKO, #A0564, 1:1,000), rabbit anti-glucagon (Phoenix, #H-028–05, 1:2,000), rabbit anti Ki67 (Abcam, #ab16667, 1:200), rabbit anti-cleaved caspase 3 (Cell Signaling, #9661, 1:200), rat anti-BrdU (Abcam, #ab6326, 1:200), mouse anti-Nkx6–1 (Hybridoma Bank, #F55A12, 1:100) rabbit anti-Mafa (Bethyl, #IHC-00352, 1:100), rabbit anti-Nkx2–2 (Sigma, #HPA003468, 1:100), rabbit anti-ALDH1A3 (Novus, #NBP2–15339, 1:100) and rabbit anti-gastrin (Millipore, #256A, 1:200). The following antibodies were used for western blot: rabbit anti-cleaved caspase 3 (Cell Signaling, #9661, 1:1,000), rabbit anti-adipsin (Santa Cruz, #sc-50419, 1:1,000), chicken anti-C3a (Abcam, #ab48581, 1:1,000), rabbit anti-Dusp26 (Invitrogen, #PA5–22013, 1:1,000), rabbit anti-actin (Cell Signaling, #8456, 1:1,000) and mouse anti-FLAG (Sigma, #A8592, 1:1,000). Recombinant C3a (R&D) was used at a concentration of 100 nM. NSC-87877 compound was dissolved in PBS and used at 20 μM concentration.

Gómez-Banoy N., Guseh J.S., Li G., Rubio-Navarro A., Chen T., Poirier B., Putzel G., Rosselot C., Pabón M.A., Camporez J.P., Bhambhani V., Hwang S.J., Yao C., Perry R.J., Mukherjee S., Larson M.G., Levy D., Dow L.E., Shulman G.I., Dephoure N., Garcia-Ocana A., Hao M., Spiegelman B.M., Ho J.E, & Lo J.C. (2019). Adipsin preserves beta cells in diabetic mice and associates with protection from type 2 diabetes in humans. Nature medicine, 25(11), 1739-1747.

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Histological and Immunohistochemical Analysis of Mouse Testes

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Mice were euthanized via cervical dislocation and testes were dissected and fixed immediately in 4% paraformaldehyde overnight at 4°C. Tissues were dehydrated in gradient ethanol, made transparent in xylen, and embedded in paraffin. Embedded tissues were cut into 5 μm sections, which were deparaffinized and rehydrated for histological analysis and immunochemistry. Hematoxylin and eosin (H&E) staining was performed for histological observation. For immunostaining, after antigen retrieval in 10 mM sodium citrate buffer, the endogenous peroxidase was cleaned up by incubating slides in 3% H₂O₂. Sections were then blocked with 5% bovine serum albumin (BSA) and incubated with primary antibody at 4°C overnight. After incubation in secondary antibody for 1 h, signals were detected using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). The following primary antibodies were used in this study: rabbit anti-Sox9 (Abcam, Cambridge, UK; ab3697), rabbit anti-Vasa (Abcam, ab13840), rabbit anti-phospho-HH3 (Abcam, ab5176), mouse anti-Sall4 (Santa Cruz, CA, USA; sc101147), rabbit anti-Vimentin (Zhongshan, Beijing, China; za0511), and rabbit anti-cleaved caspase 3 (CC3) (Cell Signaling Technology, Beverly, MA, USA; 9661).

Huang L., Wang Z.B., Qi S.T., Ma X.S., Liang Q.X., Lei G., Meng T.G., Liang L.F., Xian Y.X., Hou Y., Sun X.F., Zhao Y., Wang W.H, & Sun Q.Y. (2016). Rad9a is required for spermatogonia differentiation in mice. Oncotarget, 7(52), 86350-86358.

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Immunohistochemical Analysis of Embryonic Tissues

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Embryos were dissected, immersion fixed in 4% paraformaldehyde (PFA), and processed for immunohistochemistry using conditions previously described (Cho et al., 2012 (link)). For all antibodies, sections were incubated overnight with primary antibody at 4°C using the following dilutions: goat anti-Neogenin, 1:250 (R&D Systems), mouse anti-β-galactosidase (β-gal), 1:500 (Promega), rat anti-CD31, 1:200 (BD Pharmingen), rabbit anti-cleaved caspase-3 (CC3), 1:1000 (Cell Signaling Technology), rabbit anti-Phosphohistone H3 (PHH3), 1:1000 (EMD Millipore), mouse anti-Islet1, 1:80 (DSHB), mouse anti-Runx2, 1:5 (DSHB), rabbit anti-Col2a1, 1:350 (Origene). After rinsing in PBS, primary antibody binding was detected with the appropriate fluorescent conjugated secondary antibody (1:500) (Invitrogen). For Islet-1 and Runx2 staining, an antigen retrieval step was added before blocking where slides were incubated with 0.01M sodium citrate (pH 6.0) for five to ten minutes on a hot plate maintained at 95°C.

Quilez S., Dumontier E., Baim C., Kam J, & Cloutier J.F. (2024). Loss of Neogenin alters branchial arch development and leads to craniofacial skeletal defects. Frontiers in Cell and Developmental Biology, 12, 1256465.

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Western Blot Analysis of Protein Markers

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Proteins were separated by SDS-PAGE and immunoblotted with the following antibodies: rabbit anti-JUND (Santa Cruz, sc-74), mouse anti-ATF4 (Santa Cruz, sc-390063), rabbit anti-cleaved caspase-3 (Cell Signaling, 9664S), mouse anti-Tubulin (Sigma, T9026), mouse anti-Ran (B.D. 610340), goat anti-GFP (Abcam, 6673), mouse anti-RPL10A (Novus, 3G2), rabbit anti-RPL7 (Novus, NB100-2269), and rabbit anti-RPS6 (Abcam, ab40820).

Good A.L., Cannon C.E., Haemmerle M.W., Yang J., Stanescu D.E., Doliba N.M., Birnbaum M.J, & Stoffers D.A. (2019). JUND regulates pancreatic β cell survival during metabolic stress. Molecular Metabolism, 25, 95-106.

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Cortical Development Immunofluorescence Staining

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Perfusion, dissection, and immunofluorescence staining were conducted according to standard protocols as previously described [17 (link)]. The following are the antibodies used: mouse anti-BrdU (1:50 dilution; BD Pharmingen, Franklin Lakes, NJ, USA), rabbit anti-Cux1 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Phospho-Histone H3 (1:250 dilution; Millipore, Billerica, MA, USA), rabbit anti-Pax 6 (1:500 dilution; Covance, Princeton, NJ, USA), rabbit anti-Tbr2 (1:500 dilution; Abcam, Cambridge, UK), mouse anti-Tuj1 (1:500 dilution; Covance, Princeton, NJ, USA), rabbit anti-Gli3 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-Cleaved Caspase 3 (1:300 dilution; Cell Signaling, Madison, WI, USA).
For 5-bromo-2-deoxyuridine (BrdU, Sigma, St. Louis, MO, USA) labeling, pregnant dams were treated with 50 μg/g BrdU by intraperitoneal injection for 4 h prior to dissection at E16.5. DiI labeling was conducted by placing small crystals of the lipophilic tracer (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; Invitrogen, Waltham, MA, USA) in the neocortex to target the upper layer (2/3) and then remained in 4% paraformaldehyde (PFA). After 6 weeks, brains were sectioned at 100 μm, counterstained with bisbenzimide, mounted, and imaged.

Yabut O.R., Ng H.X., Fernandez G., Yoon K., Kuhn J, & Pleasure S.J. (2016). Loss of Suppressor of Fused in Mid-Corticogenesis Leads to the Expansion of Intermediate Progenitors. Journal of Developmental Biology, 4(4), 29.

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Immunohistochemistry and Western Blot Protocols

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For immunohistochemistry, the primary antibodies used were rabbit anti-cleaved caspase 3 (1:1000; Cat#9661, Cell Signaling Technology, Beverly, MA), mouse anti-Ki67 (1:200; Cat#556003, BD Pharmingen, Heidelberg, Germany), rat anti-BrdU (1:200; Cat#OBT0030G, Accurate Chemical), mouse anti-NeuN (1:500; Cat#MAB377, Chemicon). The secondary antibodies used were Alexa Fluor 555-conjugated goat anti-mouse IgG (1:1000; Cat#A21422, Molecular Probes), Alexa Fluor 488-conjugated goat anti-rat IgG (1:1000; Cat#A21208, Molecular Probe), Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000; Cat#4412, Cell Signaling), Alexa Fluor 555-conjugated donkey anti-goat IgG (1:1000; Cat#A21432, Molecular Probes), Alexa Fluor 647-conjugated goat anti-mouse IgG (1:1000; Cat#A21237, Molecular Probes). For western blots, the primary antibodies were, rabbit anti-p-aPKCζ/ι (T410/403)(1:500, Cat#9378, Cell Signaling), mouse anti- aPKCζ/ι (1:500; Cat#610175, BD), rabbit anti-p-CREB (S133)(1:500; Cat#9198, Cell Signaling), mouse anti- CREB (1:500; Cat#9104, Cell Signaling), rabbit anti-p300 (1:100; Cat#sc-585, Santa-Cruz), and mouse anti-p300 (1:1000, Cat#ab14984, Abcam). Secondary antibodies for western blots were HRP-conjugated goat anti-mouse or anti-rabbit (1:4000; Cat#7076 and #7074, Boehringer Mannheim).

Syal C., Seegobin M., Sarma S.N., Gouveia A., Hsu K., Niibori Y., He L., Wondisford F.E., Frankland P.W, & Wang J. (2018). Ectopic expression of aPKC-mediated phosphorylation in p300 modulates hippocampal neurogenesis, CREB binding and fear memory differently with age. Scientific Reports, 8, 13489.

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Wing Disc Immunostaining Procedure

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Wing discs of the third instar larvae were dissected, fixed, blocked, and incubated with the primary antibodies, as described [19 (link)]. After washing several times, samples were incubated with secondary antibodies in washing buffer for 2 h at room temperature and stained with 4′, 6-diamidino-2-phenylindole (DAPI). Then, samples were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA). All confocal images were acquired using Zeiss LSM710 confocal microscope and ZEN software.
Primary antibodies were used in the following dilutions: chicken anti-β-gal (ab 9361), 1:100; rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA), 1:250; rabbit anti-Dlg, 1:500 [20 (link)], mouse anti-Wg (DSHB #4D4), 1:100; mouse anti-Cut (DSHB #2B10), 1:100; guinea pig anti-Senseless (gift from Hugo Bellen), 1:1000 sheep anti-GFP (Ab direct serotec), 1:100. Secondary antibodies conjugated with Cy3 (1:200), Cy5 (1:500) and FITC (1:200) were from Alexa Flour (Jackson Immunoresearches, West Grove, PA, USA).

Kim J.Y., Tsogtbaatar O, & Cho K.O. (2021). Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster. Journal of Developmental Biology, 9(4), 43.

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Molecular Mechanisms of JAK2-FOXO3 Pathway

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Mouse anti-β-actin, mouse anti-Flag, and mouse anti-HA antibody and the following chemicals and solvents (MG132, cycloheximide, dimethyl sulfoxide (DMSO), glycerol, glycine, sodium chloride, Trizma base, and Tween20) were from Sigma (St. Louis, MO, USA). AZD1480 was from Selleckchem (Houston, TX, USA). Rabbit anti-IL4Rα, rabbit anti-IL13Rα1, mouse anti-PARP1, rabbit anti-FOXO3, mouse anti-Lamin B1, and mouse anti-GAPDH antibodies were from Santa Cruz Biotechnology. Rabbit anti-JAK2, rabbit anti-pJAK2, rabbit anti-Tyr, rabbit anti-cleaved PARP1, rabbit anti-cleaved Caspase3, rabbit anti-Bax, rabbit anti-Bim, rabbit anti-Bcl2, rabbit anti-p21, and rabbit anti-p27 antibodies were from Cell Signaling (Danvers, MA, USA). Goat anti-rabbit (111-035-003) and goat anti-mouse (115-035-003) horseradish peroxidase-conjugated IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from Genedepot (Barker, TX, USA). pECE empty/Flag-FOXO3 and pCMV3-C-HA empty/HA-JAK2 plasmid DNA were from Addgene (Watertown, MA, USA) and Sino Biological (Wayne, PA, USA), respectively.

Kang M.A., Lee J., Ha S.H., Lee C.M., Kim K.M., Jang K.Y, & Park S.H. (2019). Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways. Cancers, 11(9), 1394.

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Ovarian Protein Expression Analysis

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Twenty days after the DHEA treatment, the ovaries were immediately sampled from rats (n = 3 per group) under anesthesia and deep-frozen. Immunoblot analysis was performed as previously described [22] . The following primary antibodies were used for Immunoblot analysis: Rabbit anti-Bad (1:500, Cell Signaling Technology, Beverly, MA), mouse anticleaved caspase-9 (1:1000, Cell Signaling Technology), rabbit anticleaved caspase-3 (1:500; Cell Signaling Technology), rabbit anti-p-IκBα, rabbit anti-p–NF-κB p65 (1:1,500; Cell Signaling Technology), rabbit antinuclear factor erythroid 2-related factor 2 (Nrf2; 1:1500, Santa Cruz Biotechnology, Santa Cruz, CA), mouse antiheme oxygenase-1 (HO-1; 1:1,500; Enzo Life Sciences, Farmingdale, NY), mouse anti-NQO1 (1:1,500; Cell Signaling Technology), rabbit antihistone H3 (1:5,000; Cell Signaling Technology), rabbit anticyclooxygenase-2 (COX-2) (1:1,000, Santa Cruz Biotechnology), rabbit anti-iNOS (1:1,000, Santa Cruz Biotechnology), mouse anti-ER alpha (ERα) (1:1,000, Santa Cruz Biotechnology), and mouse anti-ERß (1:500, Santa Cruz Biotechnology) were used as primary antibodies. Immunoblot images were quantified using Image J analysis software (JAVA image processing program, NIH, Bethesda, MD).

Choi J.H., Jang M., Kim E.J., Lee M.J., Park K.S., Kim S.H., In J.G., Kwak Y.S., Park D.H., Cho S.S., Nah S.Y., Cho I.H, & Bae C.S. (2019). Korean Red Ginseng alleviates dehydroepiandrosterone-induced polycystic ovarian syndrome in rats via its antiinflammatory and antioxidant activities. Journal of Ginseng Research, 44(6), 790-798.

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