The primary antibodies used in this study were as follows: anti-BRG1 (EPNCIR111A) Abcam Cat# ab110641 (1:10,000); anti-Caspase3 (8G10), Cell Signaling Technology, Cat# 9665 (1:1000); anti-C-Caspase 3, Cell Signaling Technology, Cat# 9661 (1:1000); anti-PARP (46D11), Cell Signaling Technology, Cat# 9532 (1:1000); anti-Ki67 (B56), BD Biosciences, Cat# 550609 (1:500); anti-EpCAM (G8.8), BD Biosciences, Cat# 552370 (1:500); anti-ZO1, Invitrogen, Cat# 40-2200 (1:500); anti-8-OHd G, Abcam, Cat# ab48508 (1:500); anti-LC3A/B (D3U4C), Cell Signaling Technology, Cat# 12741 (1:1000); anti-ATG16L1 (D6A5), Cell Signaling Technology, Cat# 8089 (1:1000); anti-PCNA, Santa Cruz Biotechnology, Cat# SC-7909 (1:1000); anti-P-H3, Cell Signaling Technology, Cat# 9701 (1:1000); anti-E-Cadherin, Cell Signaling Technology, Cat# 3195T (1:1000); anti-DCLK1, Abcam, Cat# ab31704 (1:500); anti-Claudin1, Cell Signaling Technology, Cat# 4933T (1:1000); anti-ChgA, Abcam, Cat# ab715 (1:1000); anti-Hes1, Abcam, Cat# ab71559 (1:500). Raw data of immunoblotting can be found in the source data file.
Anti c caspase3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-c-caspase3 is a lab equipment product manufactured by Cell Signaling Technology. It is an antibody that specifically recognizes the cleaved form of caspase-3, a key enzyme involved in the apoptosis (programmed cell death) pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti c parp, by Cell Signaling Technology (3 mentions)
Anti gpx1, by GeneTex (2 mentions)
Anti 4 hne, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti hes1, by Abcam (1 mentions)
Anti ph3, by Cell Signaling Technology (1 mentions)
Anti parp 46d11, by Cell Signaling Technology (1 mentions)
Anti ki67 b56, by BD (1 mentions)
Anti 8 ohdg, by Abcam (1 mentions)
Anti lc3a b d3u4c, by Cell Signaling Technology (1 mentions)
Anti dclk1, by Abcam (1 mentions)
Anti claudin 1, by Cell Signaling Technology (1 mentions)
Anti chga, by Abcam (1 mentions)
Anti pcna, by Santa Cruz Biotechnology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti brg1 epncir111a, by Abcam (1 mentions)
Anti caspase 3 8g10, by Cell Signaling Technology (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
13 protocols using anti c caspase3
1
Comprehensive Antibody Characterization Protocol
2
Immunofluorescence Staining of Liver Tissue
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For immunofluorescence staining, 5μm frozen liver tissue sections were fixed with 3% PFA and blocked with species-specific serum (Sigma-Aldrich, Steinheim, Germany). Sections were incubated with specific antibodies for 1h at RT. The following antibodies were used: anti-cCaspase3 (Cell Signaling Technologies, Danvers USA), anti-CD3e (145-2C11, Becton Dickinson (BD) Biosciences, Heidelberg, Germany), anti-CD4 (RM4-5, Biolegend, San Diego, USA), anti-CD8a (53–6.7, Biolegend, San Diego, USA), anti-CD68 (AbD Serotec, Düsseldorf, Germany), anti-Ly6G and Ly6C (RB6-8C5, BD Biosciences, Heidelberg, Germany), and anti-Ki67 (MMI, Leica Biosystems, Wetzlar, Germany). Slides were washed and incubated with fluorochrome conjugated second stage antibody and DAPI (Sigma Aldrich, Steinheim, Germany) for 30min. The following second stage antibodies were used: anti-armenian-hamster-IgG-AlexaFluor®488, anti-armenian-hamster-IgG-Cy3, anti-rabbit-IgG-DyLight®549, anti-rat-IgG-DyLight®549 (all Dianova, Hamburg, Germany), and anti-rat-IgG-Alexa Fluor®488 (Life Technologies, Darmstadt, Germany). Stained sections were mounted with ProLong Gold Antifade Reagent (Invitrogen) and analyzed using an Observer.Z1 attached to an AxioCam MRm camera, an Plan-Apochromat 40x/1,3 NA and 100x/1,4 NA oil objective and the AxioVision Software (all Zeiss Microscopy).
3
Renal Morphology and Inflammation Analysis
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Changes in renal morphology were examined in paraffin-embedded tissue sections (4 μm) stained with hematoxylin. Immunohistochemical staining was performed as previously described (Xu et al., 2012 (link)). The following primary antibodies were used: anti-Wnt1 (1:100, Abcam), anti-Wnt4 (1:150, Santa Cruz Biotechnology), anti-c-caspase3 (1:500, Cell Signaling Technology), anti-F4/80 (1:200, Abcam). The secondary antibody was horseradish peroxidase-conjugated anti-mouse IgG (1:1,000; Jackson ImmnoResearch), which was used according to the manufacturer’s instructions. The peroxidase was visualized with diaminobenzidine. To determine the number of F4/80-positive cells, 8–10 fields for each mouse were examined under a microscope (magnification, 200×), and the average number of positive cells was calculated.
4
Baicalin and Mithramycin-A Apoptosis Assay
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Baicalin and mithramycin-A were procured from Sigma-Aldrich (St Louis, Missouri, USA). The annexin V-FITC and CCK-8 kits for detecting apoptosis were obtained from Beyotime (Shanghai, China), the RNAiso Plus reagent kit and the PrimeScript RT reagent kit (Perfect Real Time) were obtained from TAKARA, BIO (Kusatsu, Japan), whereas anti-sp1, anti-C-PARP, anti-C-caspase-3, and tubulin antibodies were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA).
5
Western Blot Analysis of Chondrocyte Markers
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Radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China) was used to isolate total proteins in cells, and protein concentration was quantified by a NanoDrop 3000 (Thermo Fisher Scientific). sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was used to separate proteins, and then proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. After that, membranes were blocked in skim milk for 2 h at 37°C and then incubated with primary antibody (Ab) at 4°C overnight. Following 2-h incubation with secondary Ab marked with horseradish peroxidase (HRP) (1:2000; Cell Signaling Technology, Danvers, MA, USA). The chemiluminescence was developed using an Enhanced chemiluminescence (ECL) detection kit (Beyotime). The primary Abs were as follows: anti-ADAMTS5 (1:1000; Abcam, Cambridge, UK), anti-MMP13 (1:1000; Abcam, Cambridge, UK), anti-COL2A1 (1:1000; Abcam, Cambridge, UK), anti-proliferating cell nuclear antigen (PCNA) (1:1000; Abcam, Cambridge, UK), anti-β-actin (1:4000; Cell Signaling Technology, Danvers, MA, USA), and anti-cleaved caspase-3 (anti-C-caspase3; 1:1000; Cell Signaling Technology).
6
Protein Profiling of Apoptosis Markers
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Cell lysates were extracted with the cell lysis buffer (Beyotime, China) and an Enhanced BCA Protein Assay Kit (Beyotime, China) was used to quantified the protein concentration of the cell lysates. Protein samples with 30–50 μg were loaded in each well and separated by 12% PAGE for immunoblotting. And the primary antibodies used were as follows: anti-c-PARP, anti-c-caspase3, anti-P21, anti-Aurora A, anti-Aurora B, anti-YAP, anti-phosphorylated YAP (ser127), anti-phosphorylated YAP (ser397) (Cell Signaling Technology, USA); antiβ-Actin (Kangwei, China).
7
DNA Damage Response Regulation
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DMEM, RPMI-1640 (Invitrogen, Carlsbad, CA, USA), EX527, Nicotinamide, SRT1720 (Selleckchem, Shanghai, China), Cisplatin, ADM, VP-16, Cycloheximide, MG132 (Sigma Aldrich, Shanghai, China), Puromycin (Life Technologies/Gibco), Trizol reagent (Invitrogen), anti-XRCC1, anti-Ku80, anti-SIRT1, anti- γ-H2AX, anti-c-PARP1 were purchased from Abcam, Shanghai, China, anti-Cullin 1, anti-β-TrCP, anti- c-Caspase3 were purchased from Cell Signaling Technology, Shanghai, China, anti-α-Tubulin, anti-Flag, anti-poly Ubiquitin, anti-Pan acetyl lysine, anti-HA, anti-HA agarose beads, anti-Flag beads, GST- Sepharase beads, were purchased from Sigma Aldrich, Shanghai, China.
8
Protein Expression Analysis in Cultured SGNs
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After the drug treatment, the proteins from the cultured SGNs were extracted with radioimmunoprecipitation assay buffer (RIPA) buffer (Protein Biotechnology, China). The mixture was centrifuged at 4°C and 12,000 × g in a refrigerated centrifuge and the supernatant was collected. A total of 30 μg of each protein sample was denatured and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. The primary antibodies were anti-GPX1 (1:1,000 dilution, GeneTex, GTX116040), anti-C-caspase 3 (1:500 dilution, Cell Signaling Technology, 9664), anti-4-HNE (1:1000, Abcam, ab46545), anti-phosphorylated (p)-NFκB p65 (1:1,000 dilution, Cell Signaling Technology, 3033), and anti-β-actin (1:2,000 dilution, ZSGB-BIO, TA-09). The protein signals were detected using an ECL kit (Millipore, United States) and analyzed by Image J software. The relative optical density ratio was calculated by comparison to β-actin.
9
Antibody Panel for Apoptotic Signaling
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The following antibodies were used in this study: anti-pJNK, anti-p-c-jun, anti-Bcl-xL, anti-β-gal, and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Anti-C-Caspase-3 was purchased from Cell Signalling Technology (Danvers, MA, USA). The ZAK monoclonal antibody (M02) was purchased from Abnova (Taipei, Taiwan). 3-HF was purchased from Sigma. siZAKβ was kindly provided by Dr. J. J. Yang (Chung Shan Medical University, Taichung, Taiwan).
10
Immunohistochemical Analysis of Oxidative Stress Markers
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Antibodies were purchased from the following sources: anti p53-upregulated modulator of apoptosis (PUMA), anti c-caspase 3, anti c-PARP, anti acetyl-H3, and anti acetyl-H4 (Cell Signaling Technology, Danvers, MA), anti p65 (Santa Cruz Biotech, Santa Cruz, CA), anti acetyl-p65 (Genentech, South San Francisco, CA), anti 4-hydroxynonenal (HNE) (AbCam, Cambridge, MA, USA), anti NADPH (Biorbyt, Cambridge, UK) and anti 8-hydroxy-2′-deoxyguanosine (8-OHdG) (Millipore, Milford, MA). Three-micrometer thick colon sections were rinsed in PBS, and incubated overnight at 4 °C with the appropriate primary antibodies. The colon sections were then washed with PBS, and incubated with the appropriate fluorophore-conjugated, or biotinylated secondary antibody processed with an avidin-biotin complex kit (VECTASTAIN ABC Kit; Vector Laboratories, Burlingame, CA, USA). Biotinylated secondary antibody was visualized with 3,3-diaminobenzidine-HCl (DAB). Fluorescent antibody stained slides were then treated with an anti-photo bleaching reagent and sealed with cover glass, and DAB stained slides were mounted on glass slides. Both were analyzed under an Axiovert 40 CFL microscope (Carl Zeiss AG; Oberkochen, Germany).
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