Anti cleaved caspase 3

Manufactured by BD

Sourced in Japan, United States

Anti-cleaved caspase-3 is a laboratory equipment used for the detection and quantification of cleaved caspase-3, a key enzyme involved in the apoptosis (programmed cell death) pathway. It is a reliable tool for researchers studying cell death and apoptosis-related processes.

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Lab products found in correlation

Anti insulin, by Agilent Technologies (3 mentions) Alexa fluor, by Thermo Fisher Scientific (3 mentions) Anti ctip2, by Abcam (2 mentions) Anti gfp antibody, by Nacalai Tesque (2 mentions) Anti pax6, by Fortrea (2 mentions) Anti pdx1, by R&D Systems (2 mentions) Paraformaldehyde solution, by Thermo Fisher Scientific (2 mentions) Lsm800 confocal microscope, by Zeiss (2 mentions) Anti phospho histone h3, by Merck Group (1 mentions) Anti gfap, by Merck Group (1 mentions) Anti tbr2, by Abcam (1 mentions) Anti ki 67, by Leica camera (1 mentions) Ab64693, by Abcam (1 mentions) Anti cd68, by Agilent Technologies (1 mentions) Hematoxylin gill 3, by Merck Group (1 mentions) Anti cd3, by Thermo Fisher Scientific (1 mentions) Anti cd66b, by BioLegend (1 mentions) Anti il 8, by Thermo Fisher Scientific (1 mentions) Ma5 14524, by Thermo Fisher Scientific (1 mentions) M087629 2, by Agilent Technologies (1 mentions)

10 protocols using anti cleaved caspase 3

Immunohistochemistry of Neural Development

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Immunohistochemistry was performed as described previously with slight modifications (Toda et al., 2013 (link); Kawasaki et al., 2000 (link)). Coronal sections were permeabilized with 0.3% Triton X-100/PBS and incubated overnight with primary antibodies, which included anti-Tbr2 (Abcam, UK, RRID: AB_778267), anti-Pax6 (Covance, Princeton, NJ, RRID: AB_291612), anti-Ki-67 (Leica, Germany, RRID: AB_442102), anti-phospho-histone H3 (Millipore, Billerica, MA, RRID: AB_310016), anti-phosphorylated vimentin (Medical and Biological Laboratories, Japan, RRID: AB_592963), anti-cleaved caspase 3 (BD Pharmingen, San Diego, CA, RRID: AB_397274), anti-Ctip2 (Abcam, UK, RRID: AB_2064130), anti-FOXP2 (Atlas antibodies, Sweden, RRID: AB_1078908), anti-GFAP (Sigma-Aldrich, St. Louis, MO, RRID: AB_477010) and anti-GFP antibodies (Nacalai tesque, Japan, RRID: AB_2313652; Medical and Biological Laboratories, Japan, RRID: AB_591819). After incubation with secondary antibodies and Hoechst 33342, the sections were washed and mounted.
For triple immunostaining, after double immunostaining was performed as described above, the sections were incubated with biotin-conjugated anti-phospho-histone H3 antibody (Millipore, Billerica, MA, RRID: AB_310794) and subsequently with fluorescent-dye conjugated streptavidin.

Matsumoto N., Shinmyo Y., Ichikawa Y, & Kawasaki H. (2017). Gyrification of the cerebral cortex requires FGF signaling in the mammalian brain. eLife, 6, e29285.

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Immunohistochemical Profiling of Tissue

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Formalin-fixed and paraffin-embedded tissue sections were stained with anti-CD66b (305102, Biolegend), anti-cleaved caspase-3 (559565, BD Pharmingen, Franklin Lanes, NJ, USA), M30 (12140322001, Roche, Merck, Darmstadt, Germany), anti-IL-8 (MA5-23697, Thermo Fisher Scientific), anti-CD3 (MA5-14524, Thermo Fisher Scientific), anti-CD68 (M087629-2, Dako, Agilent Technologies, Carpinteria, CA, USA) and anti-CD206 (ab64693, Abcam, Cambridge, UK) antibodies for 1 h, followed by nuclear counterstaining with Hematoxylin Gill III (1.05174.0500, Merck). See supplementary material for extended protocol.

Schimek V., Strasser K., Beer A., Göber S., Walterskirchen N., Brostjan C., Müller C., Bachleitner-Hofmann T., Bergmann M., Dolznig H, & Oehler R. (2022). Tumour cell apoptosis modulates the colorectal cancer immune microenvironment via interleukin-8-dependent neutrophil recruitment. Cell Death & Disease, 13(2), 113.

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Immunohistochemical Analysis of Grafted Kidney Cells

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Mouse kidneys with cells grafted under the capsules were washed with PBS, fixed with 4% paraformaldehyde at 4 °C overnight, and transferred to 30% sucrose solution for dehydration. The tissues were embedded in a 2:1 mixture of OCT: 30% sucrose and sectioned using a cryostat microtome. The slides were blocked and permeabilized in PBS solution containing 5% horse serum and 0.3% Triton for 1 h at RT and then incubated with primary antibodies overnight at 4 °C followed by 1 h incubation with fluorescence-conjugated secondary antibodies (Alexafluor, ThermoFisher Scientific) at RT. The following primary antibodies were used: anti-PDX1 (1:500, R&D), anti-insulin (1:500, DAKO) and anti-cleaved caspase-3 (1:1000, BD Biosciences), and anti-STEM121 (1:1000, Stem Cells Inc.). Fluorescent images were scored using MetaMorph® image analysis software (Molecular Devices).

Amin S., Cook B., Zhou T., Ghazizadeh Z., Lis R., Zhang T., Khalaj M., Crespo M., Perera M., Xiang J.Z., Zhu Z., Tomishima M., Liu C., Naji A., Evans T., Huangfu D, & Chen S. (2018). Discovery of a drug candidate for GLIS3-associated diabetes. Nature Communications, 9, 2681.

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Immunofluorescence Staining of Stem Cell Markers

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Cells were fixed in 4% paraformaldehyde solution (Affymetrix) for 20 mins, then washed three times in PBS with 5 mins incubation each. The cells were blocked and permeabilized in PBS solution containing 5% horse serum and 0.3% Triton for 1 hour at room temperature. The cells were incubated with primary antibodies overnight at 4°C, followed by three times wash in PBS with 5 mins incubation each. After 1 hour incubation with fluorescence-conjugated secondary antibodies (Alexafluor, ThermoFisher Scientific) at RT, cells were washed with PBS for three times and imaged with LSM 800 confocal microscope (Zeiss). The primary antibodies used were anti-SOX2, anti-OCT4 (1:500–1:1000 according to manufacture instructions, Cell signaling), anti-insulin (1:500, DAKO), and anti-cleaved caspase-3 (1:1000, BD Biosciences). The detailed antibody information has been included as Supplemental Table 10.

Zhao Z., D’Oliveira Albanus R., Taylor H., Tang X., Han Y., Orchard P., Varshney A., Zhang T., Manickam N., Erdos M., Narisu N., Taylor L., Saavedra X., Zhong A., Li B., Zhou T., Naji A., Liu C., Collins F., Parker S.C, & Chen S. (2023). An integrative single-cell multi-omics profiling of human pancreatic islets identifies T1D associated genes and regulatory signals. Research Square.

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Western Blot Analysis of Protein Expression

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Soluble protein extracts (50 μg) were loaded into polyacrylamide gels (9–12%) and transferred onto the nitrocellulose membranes. Membranes were immunoblotted overnight with mouse monoclonal anti-eNOS (1:2000; BD Transduction Labs), anti-peNOS (1:2000; BD Transduction Labs), anti-MMP-2 (1:2000; Millipore), anti-CD11b (1:3000; BD Biosciences), anti-HO-1 (1:3000; Stressgen), anti-IL-10 (1:1000; Abcam) and anti-cleaved caspase-3 (1:3000; BD Biosciences) antibodies. After washing, the membranes were incubated with horseradish peroxidase-linked secondary antibodies for 1 h at room temperature. Chemiluminescence enhanced bands were visualized using an automated imaging system (UVP Biospectrum). Protein levels were quantified by scanning densitometry (ImageJ Software; 1.48v, National Institutes of Health, USA).

Chen Y.C., Ho C.C., Yi C.H., Liu X.Z., Cheng T.T, & Lam C.F. (2017). Exendin-4, a glucagon-like peptide-1 analogue accelerates healing of chronic gastric ulcer in diabetic rats. PLoS ONE, 12(11), e0187434.

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Atherosclerotic Plaque Analysis Protocol

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At sacrifice, the aortic root was collected, embedded in Neg-50 (Thermo Scientific) and snap frozen in liquid nitrogen. Atherosclerotic plaque size was analyzed on Oil-Red-O (Sigma-Aldrich) stained cryosections and the necrotic core was analyzed on haematoxylin-eosin (H-E) stained sections as the acellular area with a threshold of 3000 μm².[20 (link)] Collagen content was measured by Sirius red (Sigma-Aldrich) staining. Immunohistochemical staining was performed for α-smooth muscle actin (α-SMA; Sigma-Aldrich), CD11c (Abcam) and for cleaved caspase-3 (Cell Signaling Technologies).
A shortened version of the Llewellyn protocol was applied to the spleen before staining with antibodies [21 (link)]. Cryosections were double stained with rat anti-CD68 (AbD Serotec), rat anti-Ter-119 (BD Pharmingen), rat anti-CD31 (BD Pharmingen), or Armenian hamster anti-CD11c combined with rabbit anti-cleaved caspase-3. Sections were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories) and visualized by fluorescence microscopy. All other images were acquired with Universal Grab 6.1 software using an Olympus BX40 microscope and quantified with ImageJ software (National Institutes of Health).

Rombouts M., Cools N., Grootaert M.O., de Bakker F., Van Brussel I., Wouters A., De Meyer G.R., De Winter B.Y, & Schrijvers D.M. (2017). Long-Term Depletion of Conventional Dendritic Cells Cannot Be Maintained in an Atherosclerotic Zbtb46-DTR Mouse Model. PLoS ONE, 12(1), e0169608.

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Western Blot Analysis of Apoptosis Markers

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RIPA buffer (Bio-Rad, Hercules, CA, USA) was used to extract the whole protein from HCC cells. The protein concentrations were determined by BCA assay kit (Bio-Rad). Proteins (100 μg/lane) were electrophoresed by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes (Bio-Rad). Membranes were blocked with 5% skimmed milk for one hour and then incubated with the following primary antibodies: anti-Bcl-2 (BD Biosciences), anti-Bax (BD Biosciences), anti-cleaved-caspase-3 (BD Biosciences), anti-CRABP2 (Cell Signaling Technology), anti-ERK (Abcam), anti-p-ERK (Abcam), anti-VGEF (Cell Signaling Technology), anti-VEGFR2 (Cell Signaling Technology), anti-p-VEGFR2 (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology) at 4°C overnight. The next day, the membranes were incubated with Horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Danvers, USA) for 2 h. ECL reagents were used to detect the immunoreactivity, and the protein bands were accessed by ImageJ software (National Institutes of Health, MD, USA). Relative expression levels of the indicated proteins were compared with GAPDH expression.

Chen Q., Tan L., Jin Z., Liu Y, & Zhang Z. (2020). Downregulation of CRABP2 Inhibit the Tumorigenesis of Hepatocellular Carcinoma In Vivo and In Vitro. BioMed Research International, 2020, 3098327.

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Immunocytochemistry for Pancreatic Cell Markers

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Cells were fixed in 4% paraformaldehyde solution (Affymetrix) for 20 min, then blocked and permeabilized in PBS solution containing 5% horse serum and 0.3% Triton for 1 h at room temperature. The cells were incubated with primary antibodies overnight at 4 °C followed by 1 h incubation with fluorescence-conjugated secondary antibodies (Alexafluor, ThermoFisher Scientific) at RT. For pSMAD2/3 staining, cells were permeabilized with ice-cold methanol at −20 °C for 10 min after fixation and prior to blocking. The following primary antibodies were used: anti-OCT4 (1:200, Santa Cruz), anti-SOX17 (1:500, R&D), anti-PDX1 (1:500, R&D), anti-SOX9 (1:1000, Millipore), anti-NKX6.1 (1:500, DSHB), anti-NKX2.2 (1:500, DSHB), anti-PAX6 (1:1000, Covance), anti-ISL1 (1:200, DSHB), anti-UCN3 (1:500, Pheonix Pharmaceuticals), anti-NGN3 (1:500, R&D), anti-chromogranin A (1:1000, Immunostar), anti-glucagon (1:2000, Sigma), anti-somatostatin (1:1000, DAKO), anti-ghrelin (1:500, Santa Cruz), anti-insulin (1:500, DAKO), and anti-cleaved caspase-3 (1:1000, BD Biosciences), anti-pSMAD2/3 (1:200, Cell Signaling).

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Immunofluorescence and Cytotoxicity Assay

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Cells were fixed in 4% paraformaldehyde for 10 minutes at RT, followed by permeabilization with treatment of 0.2% TritonX-100 in PBS for 10 minutes. Cells were then blocked with PBS containing 5% bovine serum albumin for 30 minutes and incubated with anti-IFNGR1, anti-IFNGR2 or anti-p62 antibodies overnight at 4 C. Then cells were incubated with anti-mouse Alexa Fluor 488 dye conjugate or anti-rabbit Alexa Fluor 594 dye conjugate for another 1 h at RT. 4',6-diamidino-2-phenylindole (DAPI) were incubated for 5 minutes at RT for nuclei staining. Cells were imaged using a confocal laser scanning microscopy (Leica SP8-DMIL) and the data were analyzed by Leica LAS AF Lite 3.3.0.
In vitro killing assay OVA-specific CD8 + CD45.1 + T cells were isolated from OT-I [C57BL/6-Tg(TcraTcrb)1100Mjb/J] mouse stain using CD90.2 microbeads (Miltenyi Biotec) and pre-activated with anti-mouse CD3/CD28 (bioxcell) for 48-72 h. LLC-OVA cells with control or Trim35 KO were plated in 96-well plate (1310 5 per well) with activated CD8 + T cells in 1:1 or 2:1 ratio in triplicates. After 6-8 h incubation, both T cells and LLC cells were collected and stained with anti-CD45.1 (eBioscience), anti-CD8a and anti-cleaved caspase3 (BD). The percentage of cleaved caspase3 + CD8 -CD45.1 -cells were analyzed using a cytometer (DxP AthenaTM V5-B5-R3) from Cytek Biosciences and further calculated using FlowJo 10.0.

Tang F., Lu C., He X., Lin W., Xie B., Gao X., Peng Y., Yang D., Sun L, & Weng L. (2023). E3 ligase Trim35 inhibits LSD1 demethylase activity through K63-linked ubiquitination and enhances anti-tumor immunity in NSCLC. Cell reports, 42(12).

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Immunohistochemistry of Neural Progenitor Markers

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Immunohistochemistry was performed as described previously with slight modifications (Kawasaki et al., 2000 (link); Toda et al., 2013 (link)). Sections were permeabilized with 0.3% Triton X-100 in PBS and blocked with 2% bovine serum albumin (BSA), 0.3% Triton X-100 in PBS. For quadruple-staining and immunostaining with Ki-67, sections were subjected to microwave antigen retrieval in a sodium citrate solution. The sections were then incubated overnight with primary antibodies, which included anti-Hop (Santa Cruz Biotechnology, RRID:AB_2687966), anti-HopX (Atlas Antibodies, RRID:AB_10603770), anti-Tbr2 (R&D Systems, RRID:AB_10569705; Abcam, RRID:AB_778267), anti-Pax6 (Millipore, RRID:AB_1587367), anti-Ki-67 (Thermo Fisher Scientific, RRID:AB_10853185), anti-cleaved caspase 3 (BD Pharmingen, RRID:AB_397274), anti-Brn2 (Santa Cruz Biotechnology, RRID:AB_2167385), anti-Ctip2 (Abcam, RRID:AB_2064130), and anti-GFP antibodies (Nacalai Tesque, RRID:AB_2313652; Medical and Biological Laboratories, RRID:AB_591819). After incubation with secondary antibodies and Hoechst 33342, the sections were washed and mounted.

Matsumoto N., Tanaka S., Horiike T., Shinmyo Y, & Kawasaki H. (2020). A discrete subtype of neural progenitor crucial for cortical folding in the gyrencephalic mammalian brain. eLife, 9, e54873.

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