The excised tissues were fixed in 4% paraformaldehyde (PFA) for 24 to 48 hours, transferred to 70% ethanol, processed, and embedded in paraffin. Sections were cut at 4 μm and mounted on slides for H&E and Periodic acid Schiff (PAS) staining. For IHC, sections were incubated with the primary antibodies for 60 minutes followed by incubation with immPRESS goat anti-rabbit or goat anti-mouse IgG polymer (catalogs MP-7602 and MP-7601, Vector BioLabs) for 30 minutes. The antigen-antibody complexes were detected using the ImmPACT DAB peroxidase (HRP) substrate (Vector BioLabs) and counterstained with CAT hematoxylin counterstain (Biocare) according to the manufacturer’s instructions. The following primary antibodies used for the IHC analysis included MAML2 TAD antibody (1:100, Bethyl IHC-00446), Ki-67 antibody (1:100, Dako, Agilent msxhu, 7240), cleaved caspase-3 antibody (1:400, Cell Signaling Technology, 9664), phospho-RB antibody (1:400, Cell Signaling Technology, 8516), Cdk4 antibody (1:300, Santa Cruz Biotechnology Inc., sc-166373), and P16 antibody (1:50, Santa Cruz Biotechnology Inc., sc-1661). TUNEL assays were performed using ApopTag Peroxidase In Situ Apoptosis Detection Kit (EMD Millipore, S7100) according to the manufacturer’s instructions. The stained tissue sections were scanned and digitized using Aperio Imagescope (Leica).
Cleaved caspase 3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, Canada, Japan, Germany, China
The Cleaved caspase-3 antibody is a laboratory reagent that detects the presence of the cleaved form of the caspase-3 protein. Caspase-3 is a key enzyme involved in the apoptosis (programmed cell death) pathway. The antibody specifically recognizes the cleaved, active form of caspase-3, which is a widely used marker for the detection of cells undergoing apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Ki 67 antibody, by Cell Signaling Technology (7 mentions)
Ripa buffer, by Thermo Fisher Scientific (7 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Caspase 3 antibody, by Cell Signaling Technology (5 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
β actin antibody, by Cell Signaling Technology (3 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (3 mentions)
Amersham hybond 0.45 pvdf membranes, by GE Healthcare (3 mentions)
Hrp conjugated anti β actin antibody, by Proteintech (3 mentions)
Clarity or clarity max western ecl blotting substrates, by Bio-Rad (3 mentions)
Dako target retrieval solution, by Agilent Technologies (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Cleaved parp antibody, by Cell Signaling Technology (3 mentions)
Sod1 antibody, by Santa Cruz Biotechnology (2 mentions)
Pierce bca assay, by Thermo Fisher Scientific (2 mentions)
Blocker d, by Roche (2 mentions)
Streptavidin hrp, by Roche (2 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (2 mentions)
Dab detection kit, by Roche (2 mentions)
161 protocols using cleaved caspase 3 antibody
1
Immunohistochemical Analysis of Tissue Samples
2
Caspase-3 Activation Assay in Cultured Cells
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Cells were grown on 4-well chamber slide (NEST Scientific, Woodbridge Township, NJ, USA) and fixed with 4% formaldehyde at room temperature for 15 min. After rinsing three times, slides were blocked by blocking buffer at room temperature for 60 min. Next, they were incubated with cleaved caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA)—conjugated Alexa Fluor 488 for 1 h at RT. Pictures were taken by Olympus (Tokyo, Japan) fluorescence microscope system, including CKX41 inverted microscope, DP74 camera and CellSense entry Software.
3
Larval Brain Staining and Imaging
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Brains from third-instar larvae were dissected for both orcein and acridine orange staining. Details of these staining protocols can be found in [57 (link)]. Preparations were analyzed under an Olympus BX51 upright microscope, or with an Olympus FV 1000 confocal microscope.
For immunohistochemistry, imaginal discs from late third instar larvae were dissected in PBS and fixed in 4% formaldehyde. Samples were washed in PBSTx and blocked in 1% BSA. Rabbit polyclonal Cleaved Caspase-3 Antibody (Cell Signaling Technology) was used in 1:200 dilution. The primary antibody was detected by Alexa Fluor 647 Goat Anti-Rabbit (Life Technologies) secondary antibody in 1:400 dilution. Pictures were taken by an Olympus FV10i confocal microscope.
For immunohistochemistry, imaginal discs from late third instar larvae were dissected in PBS and fixed in 4% formaldehyde. Samples were washed in PBSTx and blocked in 1% BSA. Rabbit polyclonal Cleaved Caspase-3 Antibody (Cell Signaling Technology) was used in 1:200 dilution. The primary antibody was detected by Alexa Fluor 647 Goat Anti-Rabbit (Life Technologies) secondary antibody in 1:400 dilution. Pictures were taken by an Olympus FV10i confocal microscope.
4
Neuronal Apoptosis Mechanism Evaluation
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The inhibitor of ER calcium-ATPase thapsigargin was purchased from Sigma–Aldrich. Neurobasal, B27 and basic fibroblast growth factor were obtained from Life Technologies. Papain was obtained from Worthington and other chemicals from Sigma–Aldrich. The Hoechst kit was from Beyotime Biotechnology Co. The ROS probe for the neuronal ROS detection was from Invitrogen. As for antibodies, the cleaved-caspase 3 antibody was from Cell Signaling Technology. The BcL-2-associated X protein (Bax), B-cell lymphoma 2 (BcL-2), 4-hydroxynonenal (HNE), and 2,4-dinitrophenol (DNP) and Pink1 antibodies were from Abcam. And anti-myc and anti-GAPDH were from Millipore. Other chemicals were of the highest purity available.
5
Quantifying Cleaved Caspase-3 and SOD1
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Cells were treated with CDN or control agents with the concentration indicated in the corresponding figure legends. Then cells were trypsinized, and cell lysates were prepared with RIPA buffer (Thermo, Catalog No. 89900). The protein concentrations were quantified by Pierce BCA assay (Thermo Scientific, Catalog No. 23250). Cleaved caspase-3 antibody was purchased from Cell Signaling Technologies (Asp175, #9661). SOD1 antibody was purchased from Santa Cruz Biotechnologies (sc-101523).
6
Radiation-Induced Gut Apoptosis Analysis
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Prap1-/- and wild-type littermate controls at 8 weeks of age received 10 G radiation (225 kV and 17.7 mA) using a RS2000 X-ray irradiator (Rad Source Technologies, Buford, GA). Weight loss was monitored daily and the mice were killed once they reached 75% of their initial body weight. Apoptotic cells were visualized in tissue sections collected 6 hours after radiation challenge using the In Situ Cell Death detection kit (Roche, Basel, Switzerland) and quantified by counting the number of positive cells per crypt in 10 random fields of view per mouse (n ≥ 9 mice) at 400× total magnification on an FV1000 confocal fluorescence microscope (Olympus). Cleaved caspase-3–positive cells were visualized 72 hours after radiation challenge using the polyclonal cleaved caspase-3 antibody from Cell Signaling (Danvers, MA). By using a total magnification of 200×, the number of positive cells in 120 random villi was enumerated for each mouse (n ≥ 6 mice), and, assuming an average of 86 enterocytes per villi, the proportion of positive cells was calculated.
7
Colorectal Cancer Mouse Model Protocol
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PD was purchased from Meilun Inc. Ki-67 antibody was purchased from BD Bioscience (USA). Cleaved Caspase-3 antibody was purchased from Cell Signaling (USA). CD31 antibody was purchased from Abcam (UK). Notch1 and cleaved Notch1 antibody was purchased from Cell Signaling Technology (USA). Azoxymethane (AOM) was purchased from Sigma (USA). Dextran sodium sulfate (DSS) was purchased from MP Biomedicals (USA). Annexin-V-FITC apoptosis detection kit and CCK-8 kit were purchased from Biotool (USA). N2 supplement, B27 supplement, EGF, noggin, Advanced DMEM/F12 and bFGF were purchased from Life Technologies (USA). CellTiter-Glo®3D cell viability detection kit was purchased from Promega (USA). k02288 (K), a highly selective inhibitor of BMP type I receptor with an IC50 of 6.4 nM for ALK6, was purchased from Selleck. Magnetic beads coated with anti-Lgr5 antibody were purchased from Miltenyi Biotec (Germany). Flow cytometer was purchased from Beckman (USA). Philips SL18 Medical X-ray accelerator was purchased from Berkshire (UK). Multifunctional microplate reader was purchased from Molecular Devices (USA). Microscope was purchased from Olympus (Japan). X-ray radiometer was purchased from Radsource.
8
Peptain-1 Delivery and Apoptosis Induction in HeLa Cells
9
Cleaved Caspase 3 Immunohistochemistry
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A rabbit polyclonal Cleaved Caspase 3 antibody (Cell Signaling) was used at 0.1 μg/mL concentration. Slides were incubated in the Discovery XT autostainer for 3 h. Incubation with secondary antibody (biotinylated goat anti-rabbit IgG; Vector labs) at a concentration of 5.75 μg/mL occurred for 20 min. Blocker D, Streptavidin—HRP, and DAB detection kit (Ventana Medical Systems, Oro Valley, AZ, USA) were used according to the manufacturer’s instructions.
10
Osteoclast Protein Phosphorylation Profiling
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To study phosphorylation and expression of proteins in osteoclasts, BMMs were induced with RANKL for different time points as described in the figures and the total cell lysates were prepared in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was determined and equal amount of protein was applied onto SDS-PAGE. After the transfer, membranes were blocked in 5% skimmed milk for 1 h at room temperature, followed by probing with specific primary antibody primary antibodies in 5% BSA in PBS-Tween (1% v/v) overnight and then washed three times with PBS-Tween (PBST) and probed with secondary antibodies from LI-COR (Odyssey Imager; donkey anti-rabbit/IRDye 800 CW/anti-goat/IRDye 800 CW anti-mouse IRDye 680 CW) for 1 h at room temperature. Membranes were then washed three times with PBST and scanned by using LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, NE, USA). The RBPJ, NEMO, IKK2, pIκB, IκB and cleaved PARP1 antibodies were purchased form Santa Cruz, Dallas, TX, USA; Cleaved Caspase 3 antibody was purchased from Cell Signaling Technology, Danvers, MA, USA; β-actin was purchased from Sigma, St Louis, MO, USA.
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