The following antibodies were used: monoclonal rabbit anti-TXNIP (#14715, Cell signaling technology, Danvers, MA, USA), monoclonal mouse anti-TXNIP (NBP1-54578, Novus Biologicals, Centennial, CO, USA), monoclonal mouse anti-OXPHOS complex antibody (ab110413, Abcam, Cambridge, UK), monoclonal rabbit anti-cleaved caspase 3 (#9661, Cell signaling technology), monoclonal mouse anti-GFAP (#3670, Cell signaling technology), monoclonal goat anti-GFAP (ab302644, Abcam), polyclonal rabbit anti-Tomm20 antibody (sc-17764, Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal rabbit anti–NF–κB p65 (D14E12) (#8242, Cell signaling technology), monoclonal rabbit anti-phospho–NF–κB p65 (Ser536) (93H1) (#3033, Cell signaling technology), monoclonal rabbit anti-NRF2 antibody (#12721, Cell signaling technology), polyclonal rabbit Histone H3 antibody (#9715, Cell signaling technology), and monoclonal mouse anti-β-actin (A5316, Sigma-Aldrich, St. Louis, MO, USA). Fluoroshield™ with DAPI (F6057, Sigma-Aldrich) was used for nuclear staining and mounting. Sections were mounted onto gelatin-coated slides with Canada Balsam (Wako, Tokyo, Japan) following dehydration.
Rabbit monoclonal anti cleaved caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit monoclonal anti-cleaved caspase-3 is a laboratory reagent used for the detection of the cleaved form of caspase-3 protein by immunoassay techniques such as Western blotting or immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti β actin, by Merck Group (2 mentions)
Rabbit monoclonal anti cleaved parp, by Cell Signaling Technology (2 mentions)
Alexa fluor 594 conjugated igg, by Thermo Fisher Scientific (2 mentions)
Monoclonal mouse anti gfap, by Cell Signaling Technology (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Polyclonal rabbit anti tomm20 antibody sc 17764, by Santa Cruz Biotechnology (1 mentions)
Polyclonal rabbit histone h3 antibody, by Cell Signaling Technology (1 mentions)
Canada balsam, by Fujifilm (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Monoclonal rat anti f4 80, by Abcam (1 mentions)
Discovery xt, by Roche (1 mentions)
Axio scan z1 digital slide scanner, by Zeiss (1 mentions)
Biotinylated goat anti rabbit, by Vector Laboratories (1 mentions)
Goat polyclonal anti hp1α, by Abcam (1 mentions)
Anti suv39h1, by Proteintech (1 mentions)
Rabbit polyclonal anti dnmt3b, by Santa Cruz Biotechnology (1 mentions)
Normal igg, by Beyotime (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Laemmli s sample buffer, by Bio-Rad (1 mentions)
Whatman, by Cytiva (1 mentions)
15 protocols using rabbit monoclonal anti cleaved caspase 3
1
Comprehensive Antibody Panel for Multimodal Analysis
2
Histological Examination of Excised Liver
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Excised liver samples were fixed in 4 % (w/v) neutrally buffered formalin, embedded in paraffin and cut into 3 mm consecutive slices for haematoxylin and eosin (H&E) and Sirius red staining as well as staining of cleaved caspase-3. Immunohistochemical stainings were performed under standardized conditions on a Discovery XT automated stainer (Ventana Medical Systems) using monoclonal rabbit anti-cleaved caspase-3 (1:250, Cell Signaling Technologies) as a primary antibody and goat anti-rabbit, biotinylated (1:750) (Vector Laboratories) as secondary antibody. Signal detection was conducted using the DiscoveryÒ DAB Map Kit (Ventana Medical Systems). For F4/80 immunoreactivity, 12 mm sections of snap frozen liver tissue were fixed for 5 min in 4 % (w/v) neutrally buffered formalin before staining with monoclonal rat anti-F4/80 (1:200, Abcam) and detecting with Dylight 550-conjugated goat anti-rat (1:100, Thermo Fisher Scientific). Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific). For the F4/80 immunostainings, 2-3 samples in each group were excluded due to insufficient sample quality. The stained tissue sections were scanned with an AxioScan.Z1 digital slide scanner (Zeiss) equipped with a 20x magnification objective.
3
Antibody Selection for CCA Research
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The following antibodies were purchased: goat polyclonal anti-HP1α (Abcam, Cambridge, MA, USA, ab77256), mouse monoclonal anti-Dicer (Abcam, ab14601), rabbit polyclonal anti-H3K9me3 (Abcam, ab8898), rabbit polyclonal anti-SFRP1 (Abcam, ab4193), mouse monoclonal anti-RNA Polymerase II (Abcam, ab817), rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA, no.9664), rabbit monoclonal anti-cleaved PARP (Cell Signaling Technology, no.5625), rabbit polyclonal anti-Dnmt1 (SantaCruz, Dallas, TX, USA, sc-20701), rabbit polyclonal anti-Dnmt3a (SantaCruz, sc-20703), rabbit polyclonal anti-Dnmt3b (SantaCruz, sc-20704), rabbit polyclonal anti-SUV39H1 (Proteintech, Wuhan, China, 10574-1-AP), mouse monoclonal anti-GST (Proteintech, 66001-1-Ig), rabbit polyclonal anti-Histone 3 (Proteintech, 17168-1-AP) and mouse monoclonal anti-β-actin (BOSTER, Wuhan, China, BM0626) were used as primary antibody. Normal IgG (Beyotime, Shanghai, China, A7007) was used as a control for co-immunoprecipitation.
Forty human CCA samples and 13 adjacent normal tissues were obtained from the Department of Biliary-Pancreatic Surgery, Tongji Hospital of Huazhong University of Science and Technology (HUST, Hubei, China). Ethics Committee at the Tongji Hospital had approved the study. Written informed consent was obtained from all patients.
Forty human CCA samples and 13 adjacent normal tissues were obtained from the Department of Biliary-Pancreatic Surgery, Tongji Hospital of Huazhong University of Science and Technology (HUST, Hubei, China). Ethics Committee at the Tongji Hospital had approved the study. Written informed consent was obtained from all patients.
4
Apoptosis Confirmation in OSCC Cells
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We assessed cleavage of PARP1 and Caspase-3 to confirm that cell death was mediated by apoptosis. OSCC cells infected with ING1 adenoviral constructs or adenoviral controls were washed with PBS, lysed in Laemmli's sample buffer (Bio-Rad, Mississauga, ON, Canada), sonicated on ice and aliquots containing 50μg of protein were loaded on 12.5% polyacrylamide gels and electrophoresis was performed at a constant 100V. Samples were transferred to nitrocellulose membrane (Whatman, Piscataway, NJ, USA) and probed with rabbit monoclonal anti-cleaved-caspase-3 (Cell Signaling Technology), mouse monoclonal anti-PARP1 and anti-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
5
Apoptotic Cell Types Identification
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Immunofluorescent double labeling was used to detect apoptotic cell types. An apoptotic marker (cleaved caspase-3) was combined with a neuronal marker (neuronal nuclear antigen, NeuN), astrocytic marker (GFAP) and microglial marker (Iba-1). The brain tissue processing methods were the same as those described above. Sections were incubated with primary antibodies, including rabbit monoclonal anti-cleaved-caspase-3 (1:200; Cell Signaling Technology), mouse monoclonal anti-rat NeuN (1:200; Millipore), mouse monoclonal anti-rat GFAP (1:200; Cell Signaling Technology), and goat polyclonal anti-Iba-1 (1:200; Novus, Centennial, USA) at 4°C overnight. After washes in phosphate buffered saline, sections were incubated with donkey anti-rabbit, donkey anti-mouse, or donkey anti-goat secondary antibodies labeled with Alexa-Fluor 488 and Alexa-Fluor 647 (1:200; Jackson ImmunoResearch) for 1 hour at room temperature. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL; Roche, Basel, Switzerland) for 10 minutes. Stained sections were covered with coverslips, and final images were acquired from the peri-lesion cortex using a fluorescence microscope (Nikon Eclipse Ti-S, Tokyo, Japan).
6
Immunofluorescence Assay of Ovarian Cancer Cells
7
Immunostaining of Proliferation and Apoptosis Markers
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Sections were washed 3 times in PBS before proceeding with immunostaining. BrdU detection required antigen retrieval (steaming at 95°C in 0.01 mol/L citrate buffer for 10 min) and incubation in 2N HCl (30 min) before antibody incubation. For all other stains (including double staining), sections underwent antigen retrieval, then incubation in 33% normal serum in PBS (1 h) and primary antibody (overnight) at room temperature in 0.3% Triton X-100 and 1% normal serum in PBS. The primary antibodies used were: mouse monoclonal anti-BrdU (1:100; Becton-Dickinson, San Jose, CA), rabbit monoclonal anti-cleaved Caspase 3 (1:300; Cell Signaling, Beverly, MA), rabbit polyclonal anti-Ki67 (1:500; Abcam, ab15580, Cambridge, England), rabbit polyclonal anti-Sox2 (1:1000; Abcam, ab97959), rabbit polyclonal anti-Tbr2 (1:300, Abcam, ab115986) and guinea pig polyclonal anti-Doublecortin (1:1000; Millipore, ab5910, Temecula, CA). For fluorescent staining, the secondary antibodies used were: Alexa Goat anti-Mouse 488, Alexa Goat anti-Rabbit 594, and Alexa Goat anti-Guinea pig 594 (Molecular Probes, Eugene, OR).
8
TRAIL-Induced Apoptosis Pathway Dissection
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Recombinant human TRAIL, pan-caspase inhibitor Z-VAD-FMK, caspase-8 specific inhibitor Z-IETD-FMK, and caspase-9 specific inhibitor Z-LEHD-FMK used in this study were purchased from R&D systems (Abingdon). TRAIL and caspase inhibitors were reconstituted according to the manufacturer's instructions. Primary antibodies used in this study include the following: rabbit monoclonal anti-cleaved PARP (Cell signaling Technology); mouse monoclonal anti-DR4 (Abcam); rabbit monoclonal anti-DR5 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-3 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-8 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-9 (Cell signaling Technology); rabbit monoclonal anti-cleaved Bid (Invitrogen); mouse monoclonal anti-HCV core (Thermo Fisher Scientific); mouse monoclonal anti-α-tubulin (Sigma); mouse monoclonal anti-β-actin (Sigma). The secondary antibodies used for Western blot analysis were HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology) and HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology).
9
Immunofluorescence Staining of Brain Sections
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Serial sagittal or coronal (40 µm) sections were cut with a cryostat and stored at −80 °C. For immunofluorescence staining, the sections were incubated overnight with primary antibody at 4 °C. The primary antibodies used were mouse monoclonal anti-CD31 (1:50; Proteintech Group, Chicago, IL, USA), sheep monoclonal anti-BrdU (1:500; Cell Signaling Technology, Danvers, MA, USA) and rabbit monoclonal anti-cleaved caspase-3 (1:500; Cell Signaling Technology, Danvers, MA, USA). The secondary antibodies used were Alexa Fluor 488-conjugated IgG (1:1000; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 594-conjugated IgG (1:1000; Invitrogen, Carlsbad, CA, USA). The nuclei of the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI).
10
Protein Expression Analysis Using Western Blot
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Specimens were lysed in a buffer supplemented with a protease inhibitor cocktail (Roche) [14 (link)]. Concentrations of proteins were measured with the Bradford assay (Bio-Rad Laboratories, Richmond, CA, USA) using a spectrophotometer. Proteins (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Billerica, MA, USA). The following primary antibodies were incubated overnight with the blocked membranes: rabbit polyclonal anti-COL2A1, rabbit polyclonal anti-MMP-13 (Santa Cruz Biotechnology), goat polyclonal anti-BMAL1, goat polyclonal anti-CLOCK, rabbit polyclonal anti-OPG, goat polyclonal anti-PER1, goat polyclonal anti-PER2, goat polyclonal anti-RANKL, mouse monoclonal anti-actin, rabbit polyclonal anti-GAPDH, and rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with the secondary antibody. Finally, specific bands were visualized with chemiluminescent reagents (Pierce Biotechnology, Rockford, IL, USA) and quantified using the ImageJ software.
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