Mouse uterine tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned into 5-μm sections, and collected on superfrost slides (Fisher Scientific, Detroit, MI). Paraffin slide sections were deparaffinized and rehydrated through a xylene/alcohol series, followed by antigen retrieval by pressure cooking on full power for 21 minutes in 10-mM citrate buffer (pH 6.0). Tissue sections were blocked in 5% normal donkey serum and incubated in primary antibody anti-CL CASP3 (1:100 dilution, #9664, Cell Signaling Technology) overnight at 4 C. Sections were washed and incubated with goat anti-rabbit biotinylated secondary antibody (1:1000) using the Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA) peroxidase goat Immunoglobulin G). Sections were stained using the AEC (3-amino-9-ethylcarbazole) horse radish peroxidase Detection Kit (Vector Laboratories, Burlingame, CA) following the manufacturer's protocol. Nuclei were counterstained blue with haemotoxylin for 2 minutes.
Anti cl casp3
Manufactured by Cell Signaling Technology
Anti-CL CASP3 is a lab equipment product offered by Cell Signaling Technology. It is an antibody that recognizes the cleaved form of caspase-3, a key enzyme involved in the execution phase of cell apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Hybond p polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti protein disulfide isomerase, by Cell Signaling Technology (1 mentions)
Hybond, by Cytiva (1 mentions)
Scn400 slide scanner, by Leica (1 mentions)
Anti p mlkl, by Abcam (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
Bond primary antibody diluent, by Leica (1 mentions)
Anti ripk3, by Abcam (1 mentions)
Anti b220, by BD (1 mentions)
Anti cd206, by Bio-Rad (1 mentions)
Bond max immunohistochemistry robot, by Leica (1 mentions)
Bx53 microscope, by Leica (1 mentions)
3 protocols using anti cl casp3
1
Immunohistochemical Analysis of Apoptosis Markers
2
Western Blot Analysis of Protein Expression
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Equivalent amounts of protein determined by bicinchoninic acid protein assay kit were resolved by NuPAGE 4%-12% Bis-Tris gel (ThermoFisher Scientific) electrophoresis and blotted to Hybond-P polyvinylidene difluoride membranes (GE Healthcare Bio-Sciences, Pittsburgh, PA). Blots were blocked with 5% nonfat dry milk in Tris-saline buffer (pH 7.4) containing 0.1% Tween-20 and then probed with the following primary antibodies: anti-CL CASP3 1:250 (#9664, Cell Signaling Technology, Danvers, MA) and anti-iPLA2 1:500 (Cat 07-169-I; Millipore, Burlington, MA). Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibody (1:5000), and the bands were visualized using an enhanced chemiluminescence detection system (ThermoFisher Scientific). The membranes were probed with anti-nuclear receptor coactivator 3 (for nuclear protein; 1:5000, #PA1-845; ThermoFisher Scientific) or anti-protein disulfide isomerase (for cytoplasmic protein; 1:1000, #3501 Cell Signaling Technology) to quantify the relative protein expression level. Images of the bands were scanned and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) for relative optical density.
3
Immunohistochemical analysis of metabolic tissues
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Paraffin sections (2 μm) were stained with haematoxylin and eosin or various primary and secondary antibodies. Paraformaldehyde (4%) fixed and paraffin embedded liver, skeletal muscle and epiWAT were incubated in Bond Primary antibody diluent (Leica) and stainings were performed on a BOND-MAX immunohistochemistry robot (Leica Biosystems) using BOND polymer refine detection solution for DAB. The following antibodies were used: anti-F4/80 (BMA Biomedicals AG, 1:120), anti-CD206 (AbD Serotec 1:200), anti-B220 (BD Biosciences; 1:3,000), anti-CD4 (eBioscience; 1:1,000), anti-cl-Casp-3 (Cell Signaling; 1:300), anti-perilipin (RDI Division of Fitzgerald, 1:1,000), anti-RIPK3 (Abcam; 1:500) and anti-p-MLKL (Abcam; 1:500). Image acquisition was performed on an Olympus BX53 microscope with a Leica SCN400 slide scanner. Stains were evaluated blinded by an experienced pathologist and inflammatory scores were determined using the following system: 0=basically no inflammation, 1≤400-fold field of view, 2=400–200-fold field of view, 3⩾200–100-fold field of view, 4=up to 40-fold field of view. The histological scoring system for non-alcoholic fatty liver disease (NAFLD) was performed according to the NAS score system63 (link).
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