For histology, organs were fixed in 4% paraformaldehyde, embedded in paraffin and cut at 5 µm thickness. Subsequently, sections were stained with hematoxylin and eosin. TUNEL assay was performed according to manufacturer’s instructions (In situ cell death detection kit, TMR red—Roche). For immunohistochemistry, sections were deparaffinized, rehydrated and antigen retrieval was done with a citrate buffer (Vector Laboratories). Sections were then incubated overnight at 4 °C with a Caspase-3 primary antibody (Cell Signaling #9661 S, 1:300) followed by a biotinylated secondary antibody (DAKO) and ABC (Vector Laboratories). Serum LDH levels were obtained at UZ-Gent (Belgium) using Cobas 8000 modular analyzer series (Roche Diagnostics, Basel, Switzerland). Serum cytokines levels were measured using a Bio-Plex Multiplex immunoassay (Bio-Rad #171304070) according to the manufacturer’s instructions.
Caspase 3 primary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Caspase-3 primary antibody is a laboratory reagent used to detect the presence of caspase-3 protein in various experimental systems. Caspase-3 is a key enzyme involved in the execution phase of apoptosis, a form of programmed cell death. This antibody can be used to study apoptotic pathways and cellular responses to various stimuli.
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Lab products found in correlation
Biotinylated secondary antibody, by Agilent Technologies (1 mentions)
Citrate buffer, by Vector Laboratories (1 mentions)
Multiplex immunoassay, by Bio-Rad (1 mentions)
Cobas 8000 modular analyzer series, by Roche (1 mentions)
Genegnome bio imaging system, by Syngene (1 mentions)
Histogel, by Thermo Fisher Scientific (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Imagexpress micro high content screening system, by Molecular Devices (1 mentions)
Goat anti rabbit igg, by Agilent Technologies (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Agilent Technologies (1 mentions)
Mayer hematoxylin solution, by Merck Group (1 mentions)
Dylight 488, by Thermo Fisher Scientific (1 mentions)
6 protocols using caspase 3 primary antibody
1
Comprehensive Histological and Biochemical Analysis
2
Quantification of Cleaved Caspase-3 in Cells
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Protein was extracted from intestinal mucosal samples and IEC-6 cells. The levels of cleaved caspase-3 expression were determined using a specific antibody against the large fragment (17 kDa) of activated caspase-3 that resulted from cleavage. Twenty micrograms of total protein was electrophoresed on a 12% (w/v) SDS-PAGE gel, transferred onto a nitrocellulose membrane and blocked with 5% (w/v) nonfat dried milk. The membranes were probed with the caspase-3 primary antibody (Cell Signaling Technology, Danvers, MA, USA, 9665, 1 : 200) followed by the peroxidase-conjugated secondary antibody. The protein signal was visualized with chemiluminescence reagents under a GeneGnome Bio Imaging System (Syngene, MD, USA). The amount of cleaved caspase-3 was quantified by densitometry and normalized to the internal control (GAPDH).
3
Caspase 3 Immunohistochemistry in Spheroids
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Immunohistochemistry (IHC) was performed as described by Wong et al. (26 (link)). Briefly, spheroids were cultured and fixed with 10% formalin, washed in 70% ethanol and embedded in HistoGel (Thermo Scientific, HG-4000-012). After fixation and HistoGel embedding, spheroids were embedded in paraffin and cut in 3 μm layer slides. Subsequently, sections were deparaffinized, dehydrated, and incubated with 1:1,000 caspase 3 primary antibody (Cell Signaling, Danvers, USA) overnight at 4°C. All slides were counterstained with eosin and coverslipped.
4
Apoptosis Detection in Cell Culture
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Cells were fixed in 4% PFA for 15 min at room temperature, then washed 3 × with PBS and blocked in 10% NDS in PBSTX. Cleaved caspase 3 primary antibody (1:250, Cell Signaling #9661) was diluted in 2% NGS and 2% BSA in PBSTX at 4 °C overnight. Cells were washed 3 × in D-PBS before incubation with secondary antibody (1:500 goat ant-rabbit 488, Invitrogen A11008) and DAPI (1:2000, Invitrogen D1306) at room temperature for 30 min. Cells were imaged in a Molecular Devices ImageXpress Micro high content screening system.
5
Immunohistochemical Detection of Caspase-3 in Liver and Kidney
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To detect caspase-3 positive cells in liver and kidney, immunohistochemistry was performed on 3 μm sections of paraffin embedded liver samples. Sections were deparaffined in a sequence of xylene, alcohol and water. As an antigen retrieval method we used for caspase-3 samples: EDTA (1 mM, pH 8.0) buffer. Next, sections were stained with Caspase-3 primary Antibody (Cell Signaling cat. nr. 9661, 100× diluted in 1 % BSA/PBS) using an indirect immunoperoxidase technique. Endogenous peroxidase was blocked using H2O2 0.3 % in phosphate-buffered saline for 30 min. After thorough washing, sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody for 30 min (Dako, Glostrup, Denmark. cat. nr. P0448), followed by rabbit anti-goat IgG as a tertiary antibody for 30 min (Dako, Glostrup, Denmark. cat. nr. P0449). The reaction was developed using DAB as chromogen and H2O2 as substrate. Sections were counterstained using Mayer hematoxylin solution (Merck, Darmstadt, Germany). Negative antibody controls were performed. Localization of immunohistochemical staining was assessed by light microscopy. For each tissue section, positive cells per field were counted by a blinded researcher in ten microscopic fields of the tissue at 10× magnification. Results were presented as number of positive cells per field.
6
Apoptosis Quantification by Caspase-3 Staining
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Apoptotic cells were detected by Caspase-3 staining and flow cytometry. MEFs were harvested, washed twice with PBS, and re-suspended in FACS buffer (1xPBS, 0.5% BSA, 0.1% sodium azide). Cells were blocked 30 min at 4 °C with blocking buffer (1xPBS containing 5% goat serum) and fixed in 100 μl fix buffer (4% formaldehyde) for 15 min at room temperature. After washing the cells with FACS buffer two times, cells were incubated with Caspase-3 primary antibody (Cat #9664, Cell signaling) diluted in IFA-Tx buffer (4% FCS, 150 nM NaCl, 10 nM HEPES, 0.1% sodium azide, 0.1% Triton X-100) for 60 min at 4 °C. After washing the cells with FACS buffer, cells were re-suspended and incubated with secondary antibody (DyLight 488, Cat# 35553, Thermo Fisher) diluted in IFA-Tx buffer for 30 min at 4 °C. Thereafter, cells were washed with 1xPBS and re-suspended in 200 μl FACS buffer for subsequent analysis using the BD LSRFortessa FACS system.
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