The rabbit anti-c-fos polyclonal antibody, rabbit anti-caspase-3 polyclonal antibody, rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) polyclonal antibody, and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Abcam (Abcam, USA). The monoclonal mouse anti-claudin-5 antibody was purchased from Invitrogen (Invitrogen, USA). The monoclonal mouse anti-matrix metalloproteinase-2 (MMP-2), monoclonal mouse anti-MMP-9, and monoclonal rabbit anti-occludin antibodies were purchased from Abcam (Abcam, USA). RIPA buffer and the BCA kit were purchased from Beyotime (Shanghai, China). The mouse IL-6 ELISA kit and the IL-1β ELISA kit were obtained from R&D Systems (Minneapolis, MN, USA).
Rabbit anti caspase 3 polyclonal antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Rabbit anti-caspase-3 polyclonal antibody is a laboratory reagent used to detect and study caspase-3 protein. Caspase-3 is a protease enzyme that plays a central role in the execution phase of cell apoptosis or programmed cell death. This antibody can be used in various immunological techniques to identify and analyze caspase-3 expression and activation.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg horseradish peroxidase, by Abcam (3 mentions)
Anti cox 2 rabbit polyclonal antibody, by Abcam (3 mentions)
Anti parp rabbit polyclonal antibody, by Abcam (2 mentions)
Anti claudin 5 mouse monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Mouse il 6 elisa kit, by R&D Systems (1 mentions)
Il 1β elisa kit, by R&D Systems (1 mentions)
Rabbit anti c fos polyclonal antibody, by Abcam (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Abbkine (1 mentions)
Boric acid h3bo3, by Sinopharm (1 mentions)
Cell total rna extraction kit, by Tiangen Biotech (1 mentions)
Bicinchoninic acid protein determination kit, by Solarbio (1 mentions)
Mouse monoclonal anti neun antibody, by Merck Group (1 mentions)
Goat anti mouse rabbit igg, by Abcam (1 mentions)
Rabbit anti β catenin polyclonal antibody, by Abcam (1 mentions)
Mouse anti β actin polyclonal antibody, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Rabbit monoclonal anti bax antibody, by Abcam (1 mentions)
10 protocols using rabbit anti caspase 3 polyclonal antibody
1
Immunohistochemistry and ELISA Analysis
2
Intestinal Epithelial Cell Apoptosis Regulation
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The following cells and reagents were used: rat small intestinal epithelial cell line IEC-6 (National Laboratory Cell Resource Sharing Platform, China), RIPM-1640 (HyClone, Logan, UT, USA), fetal bovine serum (FBS) (GIBCO, Victoria, Australia), Penicillin–streptomycin (HyClone, USA), Boric acid (H3BO3) (Cat: 20,120,524, Sinopharm Group, Beijing, China); Cell Counting Kit-8 (Abbkine, Wuhan, China), FITC Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA), PI/RNase Tillering Buffer Kit (BD Pharmingen, USA), PI3K Specific Blocker LY294002 (Selleck Chemicals, Houston, TX, USA); Akt Specific Blocker MK-2206 2HCL (Selleck Chemicals, USA), zonula occludens-1 (ZO-1) and Occludin enzyme-linked immuno sorbent assay (ELISA) kits (Dakome Technology, Shanghai, China), Cell Total RNA Extraction Kit (DP430, Tiangen, Beijing, China), Reverse Transcription Kit (Thermo Scientific, Waltham, MA, USA),; TB Green Premiere Ex Taq (Takara Bio, Kusatsu, Japan); Rabbit Anti- proliferating cell nuclear antigen (PCNA) Polyclonal Antibody, Rabbit Anti-Caspase-3 Polyclonal Antibody, Rabbit Anti-rat β-actin Antibody (Abcam, Cambridge, MA, USA), and Bicinchoninic acid Protein Determination Kit (Solarbio, Beijing, China).
3
Determining Brain Tissue Markers
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Besides HSP-70, the levels of NeuN and Caspase-3 in the brain tissue around the DBS electrodes from G1B, G2B, G3B and G4B were also determined by western-blotting. The procedures of western-blotting were similar to those mentioned above. A mouse anti-NeuN monoclonal antibody (Millipore, Billerica, MA, US) and a rabbit anti-Caspase-3 polyclonal antibody (Abcam, Cambridge, MA, US) were employed. β-actin was used as a marker to make the protein levels standardized.
4
Western Blot Analysis of Spinal Cord Injury
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The rats were severally narcotized with 10% chloral hydrate (0.33 mL/kg) via intraperitoneal injection at 3 and 5 days post-surgery, and the T8–11 spinal cord (2 mm cephalad and caudally from the epicenter) was dissected out. The tissues were homogenized in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA). Then, the concentrations of proteins were assessed using the bicinchoninic acid kit, adjusted to 2 μg/μL, and 40 μg of protein was resolved on 12% Tris-glycine SDS-PAGE gels. Then, the proteins were transferred to polyvinylidene fluoride membranes and incubated with primary antibodies (rabbit anti-β-catenin polyclonal antibody, 1:1,000, Abcam, Cambridge, UK; rabbit anti-caspase-3 polyclonal antibody, 1:1,000, Abcam; mouse anti-β-actin polyclonal antibody, 1:1,000, Abcam) at 4°C overnight after being sealed. On the following day, the membranes were washed three times with Tris-buffered saline (TBS; 150 mM NaCl, 100 mM Tris-HCl, pH 7.4) containing 0.1% Tween-20, and then incubated with goat anti-rabbit/mouse IgG (1:3,000, Abcam) at room temperature for 2 hours. Finally, the membranes were developed using a ChemiDoc-It™ TS2 Imager (UVP, LLC, Upland, CA, USA) and relative optical density was measured using ImageJ2x software (National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Immunodetection of Autophagy and Apoptosis Markers
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Proteins were electrophoresed and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with a rabbit polyclonal anti-mouse/human CDC20 antibody (1:1,000; Proteintech, Rosemont, IL, USA), a rabbit polyclonal anti-mouse/human LC3 antibody (1:1,000; Novus, Shanghai, China), a mouse monoclonal anti-human p62 antibody (1:1,000; Abcam, Cambridge, MA, USA), a rabbit monoclonal anti-human ATG5 antibody (1:1,000; Abcam, Cambridge, MA, USA), a rabbit polyclonal anti-human p-S6K antibody (1:1,000; Cell Signaling Technology, Beverly, MA, USA), a rabbit polyclonal anti-human S6K antibody (1:1,000; Cell Signaling Technology, Beverly, MA, USA), a rabbit monoclonal anti-active caspase-3 antibody (1:1000; Abcam, Cambridge, MA, USA), a rabbit polyclonal anti-caspase-3 antibody (1:1,000; Abcam, Cambridge, MA, USA), or a mouse monoclonal anti-human β-actin antibody (1:1,000; Proteintech, Wuhan, China). Membranes were then subjected to immunodetection.
6
Immunohistochemical Analysis of Apoptosis Markers
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Brain paraffin sections (4μm) were incubated overnight at 4°C with rabbit polyclonal anti-Bcl-2 antibody (1:200, Abcam, Cambridge, UK) and rabbit monoclonal anti-Bax antibody (1:200, Abcam, Cambridge, UK) respectively. After three times rinses (5 min each) with phosphate-buffered saline (PBS, pH=7.4), the sections reacted with goat anti-rabbit IgG conjugated to peroxidase secondary antibody (1:200, Aspen Biotechnology, Wuhan, China). The remaining procedures were according to the standard procedures [22] . Negative controls were established to investigate the specificity of the reactions. For semi-quantitative analysis, four visual fields (200 ×) from each section in periinfarct area were photographed under a light microscope (Olympus Corporation, Tokyo, Japan), and integrated optical density (IOD) was analyzed using Image Pro Plus 6.0 (Media Cybernetics Inc., Bethesda, MD, USA).
The immunofluorescent staining was used to assess the expression of caspase-3. Brain sections were then incubated overnight with rabbit polyclonal anti-caspase-3 antibody (1:200, Abcam, Cambridge, UK). After being rinsed with PBS, the tissues were incubated with Cy3-conjugated goat anti-rabbit IgG secondary antibody (1:50, Aspen Biotechnology, Wuhan, China). The unspecific labeling was found by replacing the primary antibody with PBS.
The immunofluorescent staining was used to assess the expression of caspase-3. Brain sections were then incubated overnight with rabbit polyclonal anti-caspase-3 antibody (1:200, Abcam, Cambridge, UK). After being rinsed with PBS, the tissues were incubated with Cy3-conjugated goat anti-rabbit IgG secondary antibody (1:50, Aspen Biotechnology, Wuhan, China). The unspecific labeling was found by replacing the primary antibody with PBS.
7
Immunohistochemical Analysis of Apoptosis Markers
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For immunohistochemistry, some sections were incubated in goat normal serum (in order to block the nonspecific site), and subsequently with anti-caspase 3 rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam, Cambridge, USA), anti-COX 2 rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam, Cambridge, USA), and anti-PARP rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam, Cambridge, USA) overnight at 4 °C. The sections were washed with PBS and then incubated with secondary antibody conjugated with horseradish peroxidase (goat anti-rabbit IgG, Abcam, Cambridge, USA) for 2 hours and detected by diaminobenzidine tetrahydrochloride for 5 minutes. Afterward, they were dehydrated and mounted. For negative controls, primary antibodies were omitted. For quantitative analysis, immunohistochemical photographs (n=5 photos from each sample were collected from all the mice in each experimental group) were assessed by densitometry using MacBiophotonics ImageJ 1.41a software on an ASUS personal computer. The data are expressed as a percentage of the total tissue area.
8
Immunohistochemical Analysis of Spinal Cord and DRG
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For immunohistochemistry, some sections were incubated with anti-caspase 3 rabbit polyclonal antibody (1:200 in PBS, v/v, Abcam), anti-COX 2 rabbit polyclonal antibody (1:200 in PBS, v/v, Abcam), and anti-S100ß rabbit polyclonal antibody (1:500 in PBS, v/v, Abcam) overnight at 4 °C. Sections were washed with PBS and incubated with secondary antibody conjugated with horseradish peroxidase (goat anti-rabbit IgG, Abcam) for 2 h. For quantitative analysis, immunohistochemical photographs (n = five photos from each five-micrometer serial transverse sections of ipsilateral spinal cord segments of the sciatic nerve and related dorsal root ganglions, the thickness of between sampled sections was 48 μm for spinal cord and 36 μm for DRG) from all rats in each experimental group were assessed by densitometry using ImageJ software. Data are expressed as a percentage of total tissue area.
9
Immunohistochemical Analysis of Apoptosis and Inflammation Markers
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For immunohistochemistry, sections were incubated in normal serum (in order to block non-specific site), and then with anti-caspase 3 rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam), anti-COX 2 rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam) and anti-PARP rabbit polyclonal antibody (1:100 in PBS, v/v, Abcam) overnight at 4 °C. Sections were washed with PBS and then incubated with secondary antibody conjugated with horseradish peroxidase (goat anti-rabbit IgG, Abcam) for 2 h and detected by diaminobenzidine tetrahydrochloride for 5 min. After wards, they were dehydrated and mounted. For negative controls, primary antibodies were omitted. For quantitative analysis at percent of total tissue area, immunohistochemical photographs (n = 5 photos from each samples collected from all mice in each experimental group) were assessed by densitometry using MacBiophotonics ImageJ 1.41a software on an ASUS personal computer. Data are expressed as a percentage of total tissue area.
10
Melanoma and Lung Cancer Cell Culture
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B16-F10 cells (murine melanoma) were obtained as a gift from Dr Hemachand Tummala. NCI-H226 cells were obtained from the NCI. Both cell lines were maintained as monolayers and passaged as necessary in an RPMI 1640 growth medium (RPMI 1640 medium supplemented with 10% Fetal bovine serum [FBS] and 1% penicillin/streptomycin). RPMI 1640 medium, penicillin/streptomycin, phosphate-buffered saline (PBS), and Dulbecco phosphate-buffered saline (DPBS) were obtained from Mediatech (Herndon, VA, USA). Fetal bovine serum was purchased from Atlanta Biologicals (Lawrenceville, GA, USA). Anti–signal transducer and activator of transcription 3 (STAT3) mouse monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti–caspase 3 rabbit polyclonal antibody was obtained from Abcam (Cambridge, MA, USA). Anti-p53 mouse monoclonal antibody was purchased from Zymed Laboratories (South San Francisco, CA, USA). Anti-p21 mouse monoclonal antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
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