For standard immunostainings, intestines were dissected in 1 × PBS (10 mM NaH2PO4/Na2HPO4, 175 mM NaCl, pH 7.4), and fixed in 4% paraformaldehyde for 25 min at room temperature. Samples were washed with 1 x PBT (0.1% Triton X‐100 in 1 × PBS) and blocked in 3% BSA in 1 × PBT for 45 min. Primary antibodies were added to the samples and incubated at 4°C overnight. The following primary antibodies were used: mouse mAb anti‐Dl (C594.9B, 1:50, developed by S. Artavanis‐Tsakonas, DSHB), mouse mAb anti‐Prospero (MR1A, 1:100, developed by Chris Doe, DSHB), rabbit anti‐pH 3 (1:2000, Millipore), rabbit anti‐active caspase 3 (1:1000, Cell Signalling), and rabbit anti‐Yun (1:1000).25 Primary antibodies were detected by fluorescent‐conjugated secondary antibodies from Jackson ImmunoResearch Laboratories. Secondary antibodies were incubated for 2 h at room temperature. DAPI (Sigma‐Aldrich; 0.1 μg/ml) was added after secondary antibody staining. The samples were mounted in mounting medium (70% glycerol containing 2.5% DABCO). All images were captured using a Zeiss inverted confocal microscope and were processed in Adobe Photoshop and Illustrator.
Rabbit anti active caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-active caspase 3 is a primary antibody that detects the active form of caspase 3, a key executioner caspase involved in apoptosis. It specifically recognizes the cleaved, active form of caspase 3.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti ph3, by Merck Group (1 mentions)
Inverted confocal microscope, by Adobe (1 mentions)
Fluorescent conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Goat anti rabbit igg hrp, by Bio-Rad (1 mentions)
Goat anti mouse igg hrp, by Bio-Rad (1 mentions)
Rabbit anti vegf, by Abcam (1 mentions)
Duramycin, by Merck Group (1 mentions)
Azithromycin, by Merck Group (1 mentions)
Mouse igg1 isotype control antibody, by R&D Systems (1 mentions)
Rabbit anti gata4 antibody, by Abcam (1 mentions)
Goat anti axl antibody, by R&D Systems (1 mentions)
Human bfgf, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Mzfliii, by Leica camera (1 mentions)
Alexa fluor 555, by Cell Signaling Technology (1 mentions)
Anti mouse cy3, by Thermo Fisher Scientific (1 mentions)
Dylight 649 donkey anti rabbit igg, by BioLegend (1 mentions)
Rabbit anti pdx1, by Abcam (1 mentions)
7 protocols using rabbit anti active caspase 3
1
Intestine Immunostaining Protocol
2
Protein Extraction and Western Blot Analysis
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Cells were washed in PBS and harvested by centrifugation. Cells were then resuspended in lysis buffer supplemented with protease inhibitors (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% Tx-100, 1 mM EGTA, 1.5 mM MgCl2, 20 mM NaF, 10 mM sodium pyrophosphate, 0.3 mM PMSF, 2.5 mM sodium orthovanadate) and incubated at 4 °C with gentle rotation for 1 h. Crude lysate was centrifuged at 17 000× g for 10 min. Equal amounts (20 μg) of protein were separated by SDS-PAGE and transferred to PVDF membrane for immunoblotting. After blocking the membrane with TBST-milk (10 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween-20, 5% non-fat powdered milk), membranes were incubated with primary antibody overnight at 4 °C followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Protein expression was detected using the ECL detection kit (GE Healthcare).
Primary antibodies used: rabbit anti-active caspase 3 (Cell Signaling), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling), rabbit anti-VEGF (Abcam).
Secondary antibodies used: goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP (Bio-Rad).
Primary antibodies used: rabbit anti-active caspase 3 (Cell Signaling), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling), rabbit anti-VEGF (Abcam).
Secondary antibodies used: goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP (Bio-Rad).
3
Antibody Procurement and Reagent Sources
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The antibodies were purchased from the following sources: Rabbit anti-GATA4 antibody (Abcam, ab84593), Goat anti-TIM1 antibody (R&D systems, AF1750), Goat anti-TIM4 antibody (R&D systems, AF2929), Goat anti-Tyro3 antibody (R&D systems, AF859), Goat anti-Axl antibody (R&D systems, AF154), Goat anti-Mer antibody (R&D systems, AF891), Mouse anti-FGF2 (EMD Millipore, 05-117), Mouse IgG1 isotype control antibody (R&D systems, MAB002), rabbit anti-active caspase-3 (Cell Signaling, #9664), mouse anti-β-actin (a3853) from Sigma Aldrich. A mouse monoclonal antibody to ZIKV NS1 protein was developed in this laboratory. The reagents were purchased from the following sources: Azithromycin (Sigma Aldrich, PZ0007), Duramycin (Sigma Aldrich, D3168), R428 (Selleckchem, S2841), human bFGF (Sigma Aldrich, F0291) and BGJ398 (Adooq Bioscience, A11159).
4
Immunohistochemical Analysis of Eye-Antennal Discs
5
Cryogenic Tissue Sectioning and Apoptosis Imaging
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Freshly collected tumors were embedded in OCT and snap frozen in liquid nitrogen. Tumors were sectioned to a thickness of 10 μm by cryotome and mounted on glass slides. Tissue sections were fixed by ice cold acetone and stained with rabbit anti active caspase 3 (Cell signaling, Danvers, MA, USA) followed by DyLight™ 649 Donkey anti-rabbit IgG (Biolegend, San Diego, CA, USA). Slides were counterstained by DAPI and were imaged on an Olympus fluorescence microscope with Metamorph acquisition software.
6
Caspase 3 Activation in Larval Wing Discs
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Third instar larvae wing discs were fixed in freshly made 4% paraformaldehyde for 15 min and washed 3 times with 1 × PBS, then stained using rabbit anti-active Caspase 3 (1 : 200) (Cell Signaling Technology, Danvers, MA, USA). Secondary antibody was anti-rabbit-Cy3 (1 : 1000, Jackson Immunochemicals, West Grove, PA, USA).
7
Quantifying Transcription Factor Localization
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In this analysis, 1 × 10 4 cells were seeded on sterilized 12-mm-diameter coverslips in 24-well plates and treated as explained above. After treatment, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min at room temperature. Slides were then permeabilized for 10 min with 0.1% Triton X-100 in PBS. Cells were blocked for 30 min with 3% bovine serum albumin, incubated at 4°C overnight with the primary antibody, and then washed and incubated for 40 min with the secondary antibodies. Images were taken with a Leica confocal microscope (Leica, Wetzlar, Germany) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) and the JACoP plug-in to determine colocalization coefficients. The following primary antibodies were used: rabbit anti-FoxO1 (1:500; Abcam, Cambridge, UK), rabbit anti-PDX-1 (1:500; Abcam), rabbit anti-MafA (1:200; Abcam), mouse anti-NeuroD (1:100; Abcam), and rabbit anti-active caspase 3 (1:100; Cell Signaling Technology, Danvers, MA) as a marker of apoptosis. Secondary antibodies were Alexa Fluor antimouse or antirabbit (1:600; Life Technologies, Carlsbad, CA). DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). Results of nuclear localization are expressed as the proportion of DAPI colocalized with each transcription factor.
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