For immunofluorescence staining, OCT-embedded frozen section (tissue) or cell climbing side (cell) was fixed with pre-cooled acetone for 10 min at RT, followed by blocking with goat serum for 1 h. Samples were incubated with primary antibodies at 4℃ overnight. These primary antibodies were used: anti-NeuN (Millipore-Chemicon), anti-CD31 (RD Systems), anti-caveolin-1, anti-glial fibrillary acidic protein (GFAP) (Abcam), anti-desmin (Abclonal), anti-cleaved caspase 3 (CST) and anti-albumin. After washing in PBS three times, the corresponding secondary antibody was subsequently used in the sample at RT for 1 h. The secondary antibodies included Alexa Fluor 594 (goat anti-rabbit) (Abcam), Alexa Fluor 488 (Goat anti-mouse) (Abcam), and Alexa Fluor 488 (Goat anti-chicken) (Invitrogen). Finally, the sample was washed and stained with diaminobenzidine/imidazole (Sigma). Immunofluorescence images were acquired by a fluorescence microscope (Nikon).
Anti cleaved caspase 3
Manufactured by ABclonal
Sourced in United Kingdom
Anti-cleaved caspase 3 is a lab equipment product that is used to detect the presence of the cleaved form of caspase 3 protein. Caspase 3 is a key enzyme involved in the apoptosis or programmed cell death process. The anti-cleaved caspase 3 product can be used in various research applications to study cell death and related biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti caspase 3, by ABclonal (2 mentions)
Anti caspase 9, by ABclonal (2 mentions)
Anti cd31, by R&D Systems (1 mentions)
Anti caveolin 1, by Abcam (1 mentions)
Anti glial fibrillary acidic protein, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Bicinchoninic acid assay system, by Beyotime (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Anti mlkl, by Abcam (1 mentions)
Anti p mlkl, by Abcam (1 mentions)
Anti cleaved parp, by ABclonal (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti parp, by ABclonal (1 mentions)
Anti c ebpβ, by Santa Cruz Biotechnology (1 mentions)
Anti cyclin b1, by Abcam (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
5 protocols using anti cleaved caspase 3
1
Immunofluorescence Staining of Tissue and Cells
2
Western Blot Analysis of Cell Signaling
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Cultured cells were treated with the indicated drugs for 48 h, followed by lysing in RIPA buffer and denaturation. Protein concentration was determined by bicinchoninic acid assay system (Beyotime). Protein sample (50 µg per lane) was separated by 12% SDS-PAGE gel, followed by electrophoretical transfer to nitrocellulose membranes. The membranes were then blocked with 5% non-fat milk for 30 min at room temperature. Primary antibodies including anti-caspase-3 (ABclonal, A2156), anti-cleaved-caspase-3 (ABclonal, A11021), anti-PARP (ABclonal, A19596), anti-cleaved-PARP (ABclonal, A19612), anti-p-MLKL (Abcam, ab196436), anti-MLKL (Abcam, ab184718), anti-N-cadherin (Abcam, ab76011), anti-E-cadherin (Abcam, ab40772), anti-Vimentin (Abcam, ab92547), anti-MMP2 (CST, 4022), anti-MMP9 (CST, 3852S), anti-TIMP1 (CST, 8946S) and anti-TIMP2 (CST, 5738S) antibodies were diluted in primary antibody dilution buffer (Coolaber, SL1360) and incubated with nitrocellulose membranes at 4°C overnight. Next, the membranes were incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase at room temperature for 2 h, followed by detection via an enhanced chemiluminescence detection kit (Thermo Fisher Scientific). GAPDH (CST, 5174S) was used as the control. Images were captured via a chemiluminescence imaging system (ChemiScope 6000 Exp).
3
Comprehensive Western Blotting Analysis of Stem Cell Proteins
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Western blotting analysis was performed as described previously23 (link). Total protein extracts from cultured HESCs were separated on 10% SDS-PAGE gels. The primary antibodies were applied according to the provided recommendations: anti-FoxM1 (1:1000, ABclonal), anti-cyclin B1 (1:1000, Abcam), anti-pH3 (1:1000, CST), anti-cleaved caspase 3 (1:1000, ABclonal), anti-caspase 3 (1:1000, ABclonal), anti-caspase 9 (1:1000, ABclonal), anti-cleaved PARP (1:1000, Bioworld), anti-STAT3 (1:1000, CST), anti-phospho-STAT3 (1:1000, CST), anti-C/EBPβ (1:1000, Santa Cruz), respectively. Anti-β-actin (1:5000, Sigma) was used as the internal control. Bands were visualized using Thermo Supersignal West Pico Chemiluminescent substrate according to the manufacturer’s instructions. The intensity of bands was determined by using Quantity One software, and the quantitative analyses of gray-scale value of each target protein vs that of individual β-actin were performed.
4
Cytotoxicity and Apoptosis Assay
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RUT (98% purity) and 5-FU (99% purity) were obtained from Aladdin, China. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), the Annexin V-FITC/PI Apoptosis Detection Kit, and dimethyl sulfoxide (DMSO) were purchased from Solarbio, Shanghai, China. The following primary antibodies were used in this study: Anti-STAT3, anti-phosphorylated (p)-STAT3 (both from Cell Signaling Technologies), anti-Bcl-2, anti-c-Myc (Affinity Biosciences), anti-cleaved caspase-3 (Abclonal), anti-CDK4 (Proteintech), and anti-GAPDH (Santa). The horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit secondary antibody used in this study was purchased from Abclonal. The electrochemiluminescence (ECL) Western blot detection reagent was acquired from Beyotime.
5
Protein Expression and Immunoblotting Analysis
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Cells were lysed with the RIPA buffer (Beyotime, Shanghai, China), containing 0.5% cocktail protease inhibitor (Roche) and 0.5 mM PMS, on a microscraper. After sonication for 15 s, the extract was centrifuged (12,000 ×g) for 15 min. Protein concentration was determined with the BCA method (standard sample: bovine serum albumin). Proteins were separated with 10% SDS-PAGE and transferred onto the PVDF membrane. After blocking with 5% skim milk in TBST for 1 h, the PVDF membrane was cultured with the anti-caspase-3 (Cat. #9668; CST, CA, USA), anti-mouse anti-EBNA1 (Cat. #sc81581; Santa Cruz, Santa Cruz, CA, USA), anti-caspase-9 (Cat. #A2636l; ABclonal, Boston, UK), anti-cleaved caspase-3 (Cat. #9664; CST), anti-p53 (Cat. #sc-126; Santa Cruz), anti-cleaved PARP-1 (Cat. No. sc-56, 196; Santa Cruz), anti-GAPDH (Cat. #10494-1-AP; Proteintech, Wuhan, Hubei, China), anti-Fas (Cat. #8023; CST), and anti-FasL (Cat. #4273; CST), respectively, at 4°C overnight. Then, the membrane was washed with TBST and then cultured with the HRP-conjugated anti-rabbit IgG (Wuhan Keri Technology) and anti-mouse IgG (Wuhan Keri Technology) secondary antibodies, respectively, for 1 h. Immunoreactivity was detected with ECL. Image J software was used to process images.
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