Fixation, processing and staining of tissue sections from tumors was carried out as previously described (18 (link)). Tumors dissected from NOD/SCID mice were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections (5μm thick) were deparaffinized followed by antigen retrieval using Citra Plus solution (Biogenex). After endogenous peroxidase inactivation, sections were incubated with primary antibodies overnight at 4°C and fluorescently labeled secondary antibodies at room temperature for 2 hrs. Sections were stained using the following antibodies: anti-Mena (1:500), anti-Ki67 (BD Biosciences), cleaved Caspase-3 (BD Biosciences). Fluorochromes on secondary antibodies included AlexaFluor 594, AlexaFluor488 and AlexaFluor 647 (Jackson Immunoresearch). Sections were mounted in Fluoromount mounting media and imaged at room temperature. Z series of images were taken on an Applied precision DeltaVision microscope using Softworx acquisition, an Olympus 40x 1.3 NA plan apo objective and a Photometrics CoolSNAP HQ camera. Images were deconvolved using Deltavision Softworx software and objective specific point spread function. At least 4 images were captured for each tumor, with at least 3 tumors per tumor group.
Cleaved caspase 3
Manufactured by BD
Sourced in United States
Cleaved caspase-3 is a laboratory reagent used in cell and molecular biology research. It is an enzyme fragment that plays a key role in the apoptosis (programmed cell death) pathway. Cleaved caspase-3 is a marker of cells undergoing apoptosis and can be used to detect and quantify this process.
Automatically generated - may contain errors
Lab products found in correlation
Anti ki67, by BD (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Ki 67 nuclear antigen, by Agilent Technologies (2 mentions)
Gapdh, by BD (2 mentions)
Caspase 3, by BD (2 mentions)
N cadherin, by BD (2 mentions)
Nanog, by BD (2 mentions)
β actin, by Merck Group (2 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
Citra plus solution, by BioGenex (1 mentions)
Alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Modfit software, by Verity Software House (1 mentions)
Rabbit anti his antibodies, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Mouse monoclonal anti human β actin, by Merck Group (1 mentions)
Monoclonal mouse anti human stat3, by BD (1 mentions)
Polyclonal rabbit anti human phosphoserine serine 727 stat3, by Cell Signaling Technology (1 mentions)
25 protocols using cleaved caspase 3
1
Immunohistochemical Analysis of Tumor Samples
2
Apoptosis and Cell Cycle Analysis in Multiple Myeloma
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Apoptosis of MM cells was detected by Annexin V binding assay using a LSR II flow cytometer (BD Biosciences, San Jose, CA). Briefly, MM cells were collected, washed twice with ice-cold PBS and once with binding buffer, and stained with FITC- or APC-conjugated Annexin V and DAPI. A minimum of 10,000 events were acquired. Data were analyzed using FlowJo software (Tree Star Inc, Ashland, OR).
For detection of cleaved caspase 3, mononuclear cells isolated from BM of patients with MM were incubated with ICG-001 for 24 hours and then collected and fixed using Cytofix/Cytoperm (BD) according to the manufacturer’s protocol. Cells were then labeled with antibodies against CD138, κ/λ light chain (both from BD), and cleaved caspase 3 (Cell Signaling Technology, Danvers, MA). MM cells were defined as CD138 and κ/λ double positive cells. Mean fluorescence intensity was determined to evaluate the level of cleaved caspase 3 in gated MM cells and surrounding non-MM CD138-negative and κ/λ-negative BM cells.
For detection of cell cycle distribution, cells were collected, fixed with 70% ethanol, and kept overnight at-20°C. Cells were then washed with PBS and stained with 1 mg/mL propidium iodide in the presence of RNase A. At least 10,000 events were acquired using LSR II flow cytometer. Data were analyzed using ModFit software (Verity Software House, Topsham, ME).
For detection of cleaved caspase 3, mononuclear cells isolated from BM of patients with MM were incubated with ICG-001 for 24 hours and then collected and fixed using Cytofix/Cytoperm (BD) according to the manufacturer’s protocol. Cells were then labeled with antibodies against CD138, κ/λ light chain (both from BD), and cleaved caspase 3 (Cell Signaling Technology, Danvers, MA). MM cells were defined as CD138 and κ/λ double positive cells. Mean fluorescence intensity was determined to evaluate the level of cleaved caspase 3 in gated MM cells and surrounding non-MM CD138-negative and κ/λ-negative BM cells.
For detection of cell cycle distribution, cells were collected, fixed with 70% ethanol, and kept overnight at-20°C. Cells were then washed with PBS and stained with 1 mg/mL propidium iodide in the presence of RNase A. At least 10,000 events were acquired using LSR II flow cytometer. Data were analyzed using ModFit software (Verity Software House, Topsham, ME).
3
Immunoblotting Analysis of Caspase Expression
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Cells were lysed in Triton lysis buffer [137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, and 20 mM Tris-HCl (pH 8.0)] containing protease inhibitors. An aliquot of each lysate was separated by SDS-PAGE using gels polymerized from 4–20% acrylamide in Tris/Glycine buffer (Invitrogen, Carlsbad, CA, USA), and immunoblotting was performed with antibodies against procaspase-3, procaspase-9 (Cell Signaling, Beverly, MA, USA), cleaved caspase-3 and procaspase-8 (BD Biosciences, San Jose, CA, USA). Eluted samples of co-immunoprecipitation experiments were also subjected to SDS-PAGE, and the electrophoresed proteins were transferred onto nitrocellulose membranes. Each membrane was incubated with mouse anti-GRP78 antibodies (BD Biosciences; 1∶1,000) or rabbit anti-His antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1∶1,000) and then with peroxidase-conjugated anti-mouse or anti-rabbit antibodies (KPL, Gaithersburg, MD, USA; 1∶5,000).
4
Western Blot Analysis of STAT Proteins
5
Immunohistochemical Analysis of MSTO-211H Xenografts
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Sections (6 mm thick) were cut from formalin-fixed paraffin-embedded MSTO-211H xenografts. Immunohistochemistry was performed using the Vectastatin Elite ABC Universal kit (Vector Laboratories, Burlingame, CA) as described previously. 20, 28 Antibodies recognizing CD31 (1:20, Dianova), Ki67 nuclear antigen (1:100; Dako), cleaved caspase-3 (1:100; BD Pharmingen), and lymphatic vessel hyaluronic acid receptor-1 (LYVE-1) (1:100) were used for our analysis. For each marker, whole-surface staining was quantified using CALOPIX Software (TRIBVIN Medical, Châtillon, France).
6
Tissue Preparation and Immunostaining Protocol
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Immunostaining was performed as described previously25 (link). Mice were deeply anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde. Fixed tissues were cryoprotected by overnight immersion in 30% sucrose, followed by embedding in OCT compound. Sections of 14 μm thickness were attached to glass slides, whereas sections of 50 μm thickness were floated on PBS. For cultures of dissociated neurons, cells were fixed by incubation in 4% paraformaldehyde for 5 min at 37 °C.
For immunostaining, tissue sections and isolated cells were permeabilized with 0.1–0.5% Triton X-100 in PBS and incubated overnight with primary antibodies, including those recognizing cleaved caspase-3 (Pharmingen), NeuN (Millipore), GFAP (SIGMA), GFP (MBL), and ChAT (Millipore). Tissues and cells were then incubated with Alexa488- and/or Cy3-conjugated secondary antibodies and 1 µg/ml Hoechst 33342, followed by washing and mounting. Epifluorescent microscopy was carried out using an Axioimager A1 microscope (Carl Zeiss, Goettingen, Germany), BZ9000 and BZ-X710 (Keyence, Japan). Confocal microscopy was performed using an LSM510 microscope (Carl Zeiss).
For immunostaining, tissue sections and isolated cells were permeabilized with 0.1–0.5% Triton X-100 in PBS and incubated overnight with primary antibodies, including those recognizing cleaved caspase-3 (Pharmingen), NeuN (Millipore), GFAP (SIGMA), GFP (MBL), and ChAT (Millipore). Tissues and cells were then incubated with Alexa488- and/or Cy3-conjugated secondary antibodies and 1 µg/ml Hoechst 33342, followed by washing and mounting. Epifluorescent microscopy was carried out using an Axioimager A1 microscope (Carl Zeiss, Goettingen, Germany), BZ9000 and BZ-X710 (Keyence, Japan). Confocal microscopy was performed using an LSM510 microscope (Carl Zeiss).
7
Western Blot Analysis of Protein Biomarkers
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Total protein was isolated from the ipsilateral hemisphere samples, and the amount of protein was measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA). The procedure of Western blotting was performed as previously described [62 (link)]. The primary antibodies were as follows: CD63 (1:1000), Tsg101 (1:1000), CD206 (1:1000) (Abcam, Shanghai, China); caspase-3 (1:1000), cleaved caspase-3 (1:1000), ARG (1:200), Notch1 (1:500), and GAPDH (1:1000) (BD Biosciences, San Jose, USA). Proteins were determined using enhanced chemiluminescence (MilliporeSigma, Burlington, USA) and photographed using a Molecular Imager VersaDoc 4000 system (Bio-Rad, Hercules, USA).
8
Protein Interaction and Expression Analysis
9
Apoptosis Quantification in Cells
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Apoptotic events were determined by Annexin V (BD Pharmingen™, #556547), cleaved caspase-3 (BD Pharmingen™, #560901) and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining. For flow cytometry, cells were harvested and stained with both Annexin V and PI for 10 min. They were then washed by PBS, and resuspended in HEPES. For cleaved caspase-3 staining, deparaffinized sections were subjected to antigen retrieval in 0.01 mol/ml citrate buffer (pH 6.0) by microwave heating. After blocking with 5% Bovine Serum Albumin (BSA, Life Technologies Inc., Carlsbad, CA, USA), the sections were incubated with rabbit monoclonal anti–cleaved caspase-3 antibody (Cell Signaling Technologies Inc., Danvers, MA, USA). TUNEL assay was performed following the manufacturer's instructions. The nuclei were counterstained using DAPI. The number of positively stained nuclei was counted from 10 fields of view and 5 different groups.
10
Shikonin, Aconitine, and Notoginsenoside R1 Bioactivities
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Shikonin, Aconitine and Notoginsenoside R1, 98% or higher purity (HPLC), were purchased from Sigma-Aldrich, Inc. (St. Louis, MO,USA) (S1 Fig ). Urethane (ethylcarbamate), lipopolysaccharide (LPS), basic fibroblast growth factor (bFGF), heparin, 12-O-tetradecanoylphorbol-13-acetate (TPA), Clodronate and Evans blue were purchased from Sigma Chemical Co. Bleomycin (BLM) from The antibodies used include: E-cadherin, N-cadherin, Vimentin, Nanog, Oct4, Snail1, CD133, Ki-67, cleaved-caspase 3, connexin 43 and fibronectin were obtained from BD Pharmingen. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG polyclonal antibody, Peroxidase substrate DAB (3′, 3′-diaminobenzidine) and AEC (3-amino-9-ethylcarbazole) were from Nichirei Bioscience (Tokyo, Japan). The mouse quantitative ELISA kits (TNF-α, MPO, ROS and 8-OHdG) were obtained from R&D Systems.
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