The following antisera were used: α-pMAD (from Ed Laufer and P. ten Dijke, at 1:2000; 51); α-dpERK (Sigma; 1:250), α-Apontic (from R. Schuh,; 52); α-Discs large (4F3; 1:50), α-DE-cadherin (DECAD2; 1:20), and anti-β-galactosidase were from Developmental Studies Hybridoma Bank. dpERK staining was carried out as described (9 ) with antibody obtained from Cell Signaling. Secondary antibodies were conjugated to Alexa-Fluor 488, 555 or 647. To assay for cell lethality, α-cleaved caspase-3 (Asp175, Cell Signaling) was used as described (53 (link)). Cell proliferation was monitored with α-phosphohistone H3 antibody (Ser 10, Cell Signaling).
α cleaved caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
α-cleaved caspase-3 is a laboratory reagent used to detect the presence and activation of the caspase-3 protein, a key mediator of apoptosis, or programmed cell death. This product represents the cleaved, active form of caspase-3.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568 goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
αphospho histone h3, by Merck Group (2 mentions)
α dperk, by Merck Group (1 mentions)
Anti β galactosidase, by Developmental Studies Hybridoma Bank (1 mentions)
Dperk, by Cell Signaling Technology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl kit, by Cytiva (1 mentions)
T5168, by Merck Group (1 mentions)
Ab172868, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Direct red 80, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Hematoxylin counterstain, by Vector Laboratories (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
α ki 67, by Cell Signaling Technology (1 mentions)
α ripk3, by Novus Biologicals (1 mentions)
13 protocols using α cleaved caspase 3
1
Immunohistochemical Analysis of Developmental Markers
2
Immunohistochemical analysis of liver tissue
3
Western Blot Analysis of Kidney Proteins
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Total proteins were isolated from frozen kidney tissue using an appropriate lysis buffer as previously described (Rodrigues-Diez et al., 2013 (link)) and quantified using a BCA protein assay kit (ThermoScientific). Proteins (50 μg) were separated on 8–15% acrylamide gels using the SDS-PAGE, as described (Rodrigues-Diez et al., 2013 (link)). Briefly, after electrophoresis, samples were transferred on to polyvinylidenedifluoride membranes (Millipore) blocked in TBS containing 0.1% Tween 20 (TBST) and 5% dry non-fat milk for 1 h at room temperature and incubated in the same buffer with different primary antibodies overnight at 4°C. After washing with TBST, membranes were incubated with the appropriate HRP (horseradish peroxidase)-conjugated secondary antibody (Invitrogen) 1 h at room temperature and developed using an ECL kit (Amersham Biosciences). Results were analyzed by LAS 4,000 and Amersham Imager 600 (GEHealthcare) and densitometered by Quantity One software (Biorad). The following primary antibodies were employed [dilution]: MLKL ([1:1,000], ab172868, abcam), α-tubulin ([1:5,000], T5168, Sigma-Aldrich) and α-Cleaved Caspase 3 ([1:1,000, #9661S, Cell Signaling). The evaluation of IL-6 in kidney tissue was done by ELISA (BD Biosciences, Cat. No. 555240) following the instructions provided by the manufacturer.
4
Quantifying Cell Proliferation and Apoptosis
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The untransduced T-47D, MDA-MB-134, HuMEC, and SUM52PE cells or the cells lentivirally transduced with the empty vector pLKO.1 or with AAMDC and MTHFD1L shRNAs were seeded onto 13 mm glass coverslips pre-treated with poly-L-lysine (Sigma-Aldrich). The adhered cells were fixed with 4% paraformaldehyde for 20 min, washed twice with PBS, blocked with 5% normal goat serum for 1 h, and separately incubated with α-Ki-67 mouse monoclonal primary antibody (Cell Signaling Technology, 1:500) and with α-cleaved caspase-3 (Cell Signaling Technology, 1:500) in antibody diluent (1% BSA and 0.3% Triton X-100 in PBS) overnight and further incubated with a goat α-mouse secondary Alexa Fluor 488-conjugated antibody (Thermo Fisher Scientific, 1:500) and goat α-rabbit secondary Alexa Fluor 488-conjugated antibody (Thermo Fisher Scientific, 1:500), respectively. The percentage of Ki-67 and cleaved caspase-3 positive cells versus total cells (Hoechst 33258, 1:5000) was assessed in at least 9 fields of view. Data were normalized to empty vector (EV) transduced cells.
5
Immunohistochemical analysis of liver tissue
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Slices of adult livers were collected and fixed in 4% PFA overnight. After dehydration, the tissues were embedded in paraffin and sectioned at 6μm. The sections were deparaffinized with xylene, rehydrated and antigens were retrieved by boiling in citrate buffer. The sections were blocked in 10% FBS and incubated with primary antibodies (αphospho-Histone H3, 06-570 Millipore; αcleaved-caspase-3, #9661 Cell Signaling) at 4°C overnight. Primary antibodies were detected with goat anti-rabbit IgG Alexa Fluor® 568 secondary antibody (A-11011, Invitrogen) and counter stained with DAPI. Images were acquired using confocal microscopy.
6
Histological Assessment of NAFLD and Fibrosis
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Livers were sectioned, formalin-fixed paraffin-embedded (FFPE) and stained with hematoxylin-eosin (H&E) for assessing NAFLD activity score (NAS), as described. 13 (link) To assess fibrosis, deparaffinized and rehydrated slides were stained with 0.1% Sirius Red stain (Direct Red 80, Sigma-Aldrich, St Louis, MO) and imaged by Nikon Eclipse 90i Microscope (Nikon, Melville, NY), as described. 9 (link) For immunohistochemical analysis, FFPE sections were deparaffinized in xylene, progressively rehydrated in alcohol gradient before quenching peroxidases with 3% H 2 O 2 and . heat-induced antigen-retrieval using 10mM sodium citrate buffer (pH 6.0) or Tris-buffer (pH 10.0), per antibody requirement. The Abcam antibodies [α-CD68 (1:150), Mac2 (1:250), MPO (1:1000), CD4 (1:1000), CD8 (1:2000), and α-Ki67 (1:100, monoclonal)] and α-Cleaved Caspase 3 (1:200, Cell Signaling, Danvers, MA) were used. This was followed by blotting with ImmPRESS HRP horse anti-mouse or anti-rabbit (Vector labs, Newark, CA) for 30min before being treated with 3,3'-Diaminobenzidine (DAB, Vector labs) and hematoxylin counterstain (Vector labs). Sections were evaluated blindly and positively stained cells were counted in 5 fields/mouse at 40X magnification.
7
Liver Protein Expression Analysis
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Protein lysates were isolated from liver samples as described previously [9 (link)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane and analyzed by immunoblot with the following antibodies: α-NEMO, α-β-actin (Sigma Aldrich, St. Louis, MO, USA), α-cleaved Caspase-3, α-cleaved Caspase-8, α-IKKβ, p-JNK, JNK1 (Cell Signaling Technology, Danvers, MA, USA), α-IKKα, α-RIPK3 (Novus Biologicals, Centennials, CO, USA), α-PCNA (Thermo Fisher Scientific, Waltham, MA, USA), and α-GAPDH (Bio-Rad Laborories, Hercules, CA, USA). As secondary antibodies, α-rabbit-HRP, and α-mouse-HRP (GE Healthcare, Little Chalfont, UK) were used. Densitometry was performed by using ImageJ software (ImageJ, NIH, Bethesda, MD, USA).
8
Immunofluorescence Staining of Mouse Brain
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Mice were anesthetized with a mix of ketamine/xylacine and perfused with 4% paraformaldehyde in PBS. After an overnight postfixation, coronal vibratome sections (50 μm) were obtained, washed in PBS and PBS − 0.1% Triton X-100 (PBT) and incubated for 1 h at room temperature with 3-5% heat inactivated newborn calf serum in PBT. The primary antibodies used were α-Kdm1a (ab17721, 1:200 or 1:1000), α-NeuN (Chemicon MAB377, 1:500), α-GFAP (G3893, Sigma, 1:500), α-Cleaved Caspase-3 (9661, Cell Signaling, 1:500), α-Fos (226004, Synaptic Systems, 1:500), α-Parvalbumin (P3088, Sigma-Aldrich, 1:500), α-Pecam 1 (550274, BD Pharmingen™, 1:500), α-H3K27me3 (07-449, Millipore, 1:500), α-H3K27me3 (ab6002, Abcam, 1:250) and α-H3K27ac (ab4729, Abcam, 1:500). Nuclei were counterstained with a 1 nM DAPI solution (Invitrogen). For the TUNEL assay, we used the in situ Cell Death Detection Kit (11684795910, Roche) following the manufacturer’s instructions. DAB staining was performed according to the instructions of the product (11718096001, Roche). Images were taken with an Inverted Confocal Microscope Olympus FV1200 and Zeiss LSM 880.
9
Evaluating Apoptosis Signaling Pathways
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Cells were collected 48 h after infection with adenovirus, and total cell lysates were prepared using RIPA buffer supplemented with protease inhibitors. Protein concentration in samples was determined using the DC Protein Assay (Bio-Rad) as per the manufacturer's protocol. Equal amounts of total protein from samples were denatured by boiling in Laemmli buffer, resolved using SDS-PAGE and transferred onto nitrocellulose membrane (Millipore, Darmstadt, Germany). The expression of cleaved caspase-3 and PARP-1 (cleaved and uncleaved) fragments was determined using α-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA) and α-PARP-1 (Santa Cruz) primary antibodies. To quantify loading of samples, all membranes were stripped and re-blotted with anti-β actin antibodies (Santa Cruz).
10
Immunohistochemical Analysis of Liver Tissue
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Liver tissue was fixed in 4% paraformaldehyde and paraffin embedded. Paraffin sections (2 μm) were stained with hematoxylin and eosin (H&E) or various primary and secondary antibodies. Automated immunohistochemical stainings, image acquisition and quantification were performed as previously described [9 (link)]. The following antibodies were used: α-pan-Cytokeratin (Dako A/S; 1:300), α-Ki67 (NeoMarkers, Fremont, CA, USA; 1:200), α-cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA; 1:300), and F4/80 (BMA Biomedicals AG, 1:120, Augst, Switzerland).
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