Total protein of CSCC tissues and cells were collected with RIPA buffer (Sigma-Aldrich; Merck KGaA). Protein concentrations were determined with a BCA assay (Bio-Rad Laboratories, Inc.). Then, a total of 20 µg protein per lane was transferred to the nitrocellulose membrane after 10% SDS-PAGE and blocked by 5% TBST (0.1% Tween-20) for 1 h at room temperature. The membranes were incubated with rabbit anti-DDX46 (1:1,000; cat. no. ab72083; Abcam), rabbit anti-Bcl-2 antibody (1:1500; cat. no. ab59348; Abcam), rabbit anti-Survivin antibody (1:1,000; cat. no. ab469; Abcam), rabbit anti-Bax antibody (1:1,000; cat. no. ab53154; Abcam), rabbit anti-Cleaved Caspase-3 antibody (1:1,000; cat. no. ab2302; Abcam), rabbit anti-Beclin 1 antibody (ab62557) (1:1,000; cat. no. ab62557; Abcam), rabbit anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) antibody (1:1,000; cat. no. ab128025; Abcam) and anti-β-actin (1:2,000; cat. no. ab8227; Abcam) at 4°C overnight. The PVDF membrane was washed three times with TBST and incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. ab97051; Abcam). Blot bands were visualized by ECL systems (Pierce; Thermo Fisher Scientific, Inc.). Densitometry was performed by using ImageJ v1.47 software (National Institutes of Health) (10 (link)).
Rabbit anti cleaved caspase 3 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Rabbit anti-cleaved caspase-3 antibody is a laboratory tool used for the detection and analysis of cleaved caspase-3, a key regulatory protein involved in the apoptosis (programmed cell death) pathway. This antibody specifically recognizes the cleaved form of caspase-3, which is a hallmark of cells undergoing apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti bcl 2 antibody, by Abcam (1 mentions)
Rabbit anti bax antibody, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bca assay, by Bio-Rad (1 mentions)
Anti β actin, by Abcam (1 mentions)
P bad, by Abcam (1 mentions)
Rabbit anti bad, by Abcam (1 mentions)
Ly294002, by Merck Group (1 mentions)
P mtor, by Abcam (1 mentions)
Rabbit anti claudin 5 antibody, by Abcam (1 mentions)
Biotinylated anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Avidin biotin peroxidase complex, by Vector Laboratories (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Sh sy5y, by American Type Culture Collection (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
6 protocols using rabbit anti cleaved caspase 3 antibody
1
Protein Expression Analysis in CSCC
2
Investigating Neurochemical Signaling Pathways
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We obtained glycine, Tris, TritonX-100, DA, homovanillic acid (HVA), 3, 4-dihydroxyphenylacetic acid (DOPAC), MPTP, dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and LY294002 from Sigma-Aldrich (St. Louis, MO, United States). From our institutional pharmacy, this study acquired Madopar (Shanghai Roche Led., Shanghai, China). Abcam (Dako, Cambridgeshire, United Kingdom) offered Rat anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-Akt (total) antibody, rabbit anti-BAD, rabbit anti-cleaved-Caspase-3 antibody, rabbit anti-phospho-Akt (Ser473) antibody, p-BAD, mTOR, p-mTOR, BDNF, CREB, and p-CREB antibodies, horseradish peroxidase (HRP)-conjugated anti-rabbit, and HRP-conjugated rat antibodies. All other chemicals exhibited great-purity analysis level and originated in Shanghai Chemical Reagent Co., Ltd. (Shanghai, China).
3
Protein Analysis of Brain Damage
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Briefly, the total proteins from the damaged brain tissue or blood were extracted by centrifugation at 4°C for 10 minutes. Then, the proteins were separated by SDS-polyacrylamide gel electrophoresis onto the nitrocellulose membrane and buffered and blocked with skimmed milk powder at room temperature for 1 h. The membranes were incubated with the rabbit anti-irisin antibody (1 : 1000; Abcam), the rabbit anti-occludin antibody (1 : 1000; CST), the rabbit anti-claudin-5 antibody (1 : 1000; Abcam), the rabbit anti-zonula occludens-1 (ZO-1) antibody (1 : 1000; CST), the rabbit anti-nuclear factor E2-related factor 2 (Nrf2) antibody (1 : 1000; CST), the mouse anti-quinine oxidoreductase (NQO-1) antibody (1 : 1000; CST), the rabbit anti-hemeoxygenase-1 (HO-1) antibody (1 : 1000; CST), the rabbit anti-cytochrome C (Cyt-C) antibody (1 : 1000; CST), the rabbit anti-cytochrome C oxidase (COX IV) antibody (1 : 1000; CST), the rabbit anti-BCL2-associated X protein (Bax) antibody (1 : 1000; CST), the rabbit anti-cleaved caspase-3 antibody (1 : 1000; Abcam), and the rabbit anti-uncoupling protein 2 (UCP2) antibody (1 : 1000; CST) at 4°C overnight. After being washed with TBST, the membranes were incubated with the secondary anti-rabbit or anti-mouse IgGs (1 : 50000; KPL) at room temperature for 30 min. Optical density analysis for quantification was performed on the ImageJ software (NIH).
4
Myocardial CEACAM1 and Caspase-3 Expression
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To detect expression of CEACAM1 and cleaved caspase-3 in the myocardium, sections of the middle LV slice were incubated overnight at 4 °C with rabbit anti-CEACAM1 antibody (Santa Cruz Biotechnology, Dallas, USA) or rabbit anti-cleaved caspase-3 antibody (Abcam, Cambridge, UK). Non-immunized rabbit IgG was used as the negative control. Then the sections were incubated with biotinylated anti-rabbit IgG and covered with streptavid in peroxidase, after which peroxidase activity was visualized with 3,3′-diaminobenzidine tetrahydrochloride.
5
Immunohistochemical Analysis of Apoptosis
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Immunohistochemistry for cleaved caspase-3-positive cells and cleaved caspase-9-positive cells was done, like the method described below [13 (link)]. The paraffin slides containing Achilles tendon tissues were deparaffinized by xylene, treated with ethanol, and rehydrated by water for 5 minutes. The Achilles tendon tissues were boiled in 10mM citric acid (pH, 6.0) for 10 minutes and treated with rabbit anticleaved caspase-3 antibody or rabbit anticleaved caspase-9 antibody (Abcam, Cambridge, UK) overnight at a dilution of 1:200. The sections were treated with biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 hour and then treated with avidinbiotin-peroxidase complex (Vector Laboratories) at room temperature for 1 hour. The sections were treated with 0.05% 3,3’-diaminobenzidine and 0.01% H2O2 in 50mM Tris-buffer (pH, 7.6) for 3 minutes for visualization of immunoreactivity. After air-drying the slides at room temperature overnight, Permount (Fisher Scientific) was finally used to mount the coverslips.
6
Investigating Neurogenesis Modulation
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Purchase human neurocytoma cell SH-SY5Y from American Type Culture Collection; MPP + was purchased from Sigma in the United States; Ginsenoside Rg5 (CAS No. 186763-78-0, purity ≥98 %, specification 10 mg/bottle) was purchased from Shanghai Yuanye Biological; Purchase Cell Counting Kit (CCK-8) from Tongren Institute of Biology, Japan; Purchase Annexin-Fluorescein V-FITC (Annexin V-FITC) cell apoptosis detection kit from Shanghai Yubo Biological Company; Lipofectamine 2000, Trizol lysate, Radioimmunoprecipitation Assay (RIPA) lysate were purchased from Invitrogen, USA; miR-874-3p mimics, miR-874-3p inhibitors (anti-miR-874-3p), TXNIP small interfering RNA (si-TXNIP) and corresponding controls (miR-con, anti-miR-con, si-con) and dual luciferase reporter gene vector were provided by Guangzhou Ruibo Biological Company; Purchase rabbit anti-TXNIP antibody, rabbit anticleaved-caspase-3 antibody, rabbit anti-beta (β)-actin antibody and goat anti-rabbit IgG from Abcam, USA.
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