Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco (USA). Fetal bovine serum (FBS) was from Hyclone Lab Inc. (USA). Sulforhodamine B (SRB), Hoechst 33,258, 2′,7′-dichlorodihydrofluorescin (DCHF) and JC-1 were from Sigma Co. (USA). Primary antibodies against β-actin, PI3K and p-PI3K were from Santa Cruz Biotechnology (USA). Primary antibody against caspase 3, PARP, LC3B, p70S6K, p-p70S6K, p-mTOR, mTOR, Akt, p-Akt, p38 MAPK, p-p38 MAPK and secondary antibody (horseradish peroxidase) were from Cell Signaling Technology (USA). Primary antibody against p62 was from BD Transduction Laboratories. Primary antibody against LOX-1 was from ABclonal (USA). Wortmannin was obtained from Selleck (USA). Ox-LDL was purchased from Beijing Xiesheng Biotechnology (China). All other reagents were ultrapure grade.
Primary antibody against caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
The primary antibody against caspase-3 is a laboratory reagent used to detect the presence and expression of the caspase-3 protein in cells and tissues. Caspase-3 is a key enzyme involved in the execution phase of apoptosis, or programmed cell death. This antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to study the regulation and activity of caspase-3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Wortmannin, by Selleck Chemicals (1 mentions)
P p38 mapk, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
P pi3k, by Santa Cruz Biotechnology (1 mentions)
2 7 dichlorodihydrofluorescin dchf, by Merck Group (1 mentions)
Primary antibodies against β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase secondary antibody, by Cell Signaling Technology (1 mentions)
Primary antibody against p62, by BD (1 mentions)
Sulforhodamine b srb, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Bx microscope, by Olympus (1 mentions)
5 5 dimethyl 1 pyrroline n oxide, by Merck Group (1 mentions)
6 protocols using primary antibody against caspase 3
1
Cytotoxicity Assays and Apoptosis Analysis
2
Caspase-3 Protein Expression Assay
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A549 cells treated with HPRP-A1 with or without iRGD were washed with ice-cold PBS and solubilized in RIPA lysis buffer. After incubating on ice for 2 h, the insoluble materials were removed by centrifugation at 12000 rpm for 15 min at 4 °C. Accurate protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit. Protein samples (80 μg) were separated on 12% SDS-PAGE gels and then transferred to polyvinylidene fluoride membranes. Membranes were blocked for 2.5 h with 5% skim milk in PBS at room temperature and blotted with the primary antibody against caspase-3 (1:5000 dilution; Cell Signaling Technology, MA, US 9665)42 (link) overnight at 4 °C. β-actin antibody (Santa Cruz Biotechnology, CA, US. sc-4778) was used at 1:1,000 dilution as a loading control. After washing by PBS with Tween-20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (abcam, MA, US) for 2 h at room temperature. The blots were detected using an enhanced chemiluminescence kit (Millipore, USA). The results were visualized on the Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd., Beijing, China)43 (link).
3
Immunohistochemistry Analysis of Caspase-3 in Ischemia/Reperfusion Injury
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For IHC staining, liver tissues collected after ischemia/reperfusion injury in mice were fixed with 10% neutral formalin for 24 h at room temperature and then embedded in paraffin. Samples were sectioned at a thickness of 4 µm, and the sections were dewaxed and dehydrated. After antigen retrieval in citrate buffer at 95°C for 15 min, the sections were blocked with 10% bovine serum albumin (cat. no. A1933; Sigma-Aldrich; Merck KGaA) overnight at 4°C. Then, the sections were incubated with primary antibody against caspase-3 (cat. no. 9662; Cell Signaling Technology, Inc.; 1:100) at 4°C overnight. Subsequently, the sections were incubated with goat Anti-Rabbit IgG H&L (Alexa Fluor® 488; cat. no. ab150081; 1:2,000; Abcam) for 30 min at 37°C. Finally, the immune-reactivity was visualized by staining with diaminobenzidine (DAB; cat. no. D-5637; Sigma-Aldrich; Merck KGaA) at room temperature for 3 min and analyzed using a light microscope (Olympus BX microscope; magnification, ×200).
4
Antioxidant and Apoptosis Assays
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Chlorogenic acid (CGA) was provided by Professor Sam Sik Kang (Seoul National University, Seoul, Korea) (Lee et al., 2010 ). The following compounds were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA): 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, N-acetyl cysteine (NAC), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] bromide (MTT), and Hoechst 33342 dye. Primary antibodies against Bcl-2 and Bax were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); a primary antibody against actin was purchased from Sigma-Aldrich Inc.; and a primary antibody against caspase-3 was purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals and reagents were of analytical grade.
5
Quantification of Cleaved Caspase-3 in Macrophages
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Extraction of total protein of human macrophages and measurement of protein concentration were described above. Protein samples of 40 μg were separated in 10% SDS-PAGE and then electrically transferred onto nitrocellulose membrane. Then the membrane was blocked in 5% skim milk for 1 h and incubated with primary antibody against cleaved caspase-3 and primary antibody against caspase-3 (Cell Signal Technology, Boston, MA, USA) at 4 °C overnight. HRP-conjugated secondary antibody was added and incubated at room temperature for 1 h after washing. Immunoreactions were visualized by X-ray film (Eastman Kodak Company, Rochester, NY, USA) exposure after the electrochemical luminescence (ECL).
6
Caspase-3 and Hsp70 Expression in U-937 Cells
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Cultured U-937wt cells were treated with BT44 at a concentration of 50 μM, then 2 μM of etoposide was administered 6 h later. After 15 h of incubation, cells were lysed in RIPA buffer, separated by PAGE on a 15% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with PBS containing 5% (w/v) skimmed milk and incubated with a primary antibody against Caspase-3 (Cell Signaling, Danvers, MA, USA) and secondary antibodies (Abcam, Cambridge, UK) at room temperature for 1 h. Band intensity was quantified using the ChemiDocTM system (Bio-Rad, Hercules, CA, USA). To assess the content of Hsp70 in treated cells, 20 μg of the same cell lysates were separated by PAGE on an 11.5% gel. The membrane was probed with an anti-Hsp70 monoclonal antibody (Clone 2H9) followed by a secondary anti-mouse antibody (Abcam).
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