The wing imaginal discs and pupal retinas were dissected from late third instar larvae and twenty-four hours after puparium formation (APF) pupae, respectively, then fixed for 17 min in phosphate-buffered saline (PBS) with 4% paraformaldehyde and then blocked in PBS with 0.3% Triton X and 5% BSA (bovine serum albumin) for 30 min at room temperature and incubated with primary antibodies overnight at 4°C. After washed with PBS-Triton X three times, the wing discs were incubated with secondary antibody for 1 h at room temperature. The stained wing discs were then mounted on the slides with glycerol. Primary antibodies: mouse anti-Cut (2B10, 1:200, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA), mouse anti-Delta (C594.B9, 1:200, DSHB), mouse anti-Notch (C17.9C6, 1:100, DSHB), mouse anti-β-gal (1:1000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-cleaved caspase-3 (1:200; Abcam). Secondary antibodies [anti-mouse Cy3 (1:1000), anti- rabbit Cy5 (1:1000)] were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The fluorescent images were acquired by confocal microscope TCS SP5 (Imaging Core, First Core Labs, National Taiwan University College of Medicine) and Axio Imager A1 Microscope (Zeiss, Thornwood, NY, USA). Leica LAS AF Lite and Adobe Photoshop were used to analyze and process images.
Rabbit anti cleaved caspase 3
Manufactured by Abcam
Sourced in United States, United Kingdom
Rabbit anti-cleaved caspase-3 is a primary antibody that recognizes the cleaved form of caspase-3, a key executioner protease involved in apoptosis. This antibody can be used to detect and analyze apoptosis in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti bax, by Abcam (5 mentions)
Rabbit anti bcl 2, by Abcam (4 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Mouse anti bcl 2, by Abcam (3 mentions)
Mouse anti bax, by Abcam (3 mentions)
Anti gapdh antibody, by BioLegend (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Anti human cd44 biotin, by Thermo Fisher Scientific (3 mentions)
Phycoerythrin pe anti human cd44, by Thermo Fisher Scientific (3 mentions)
Uorescein isothiocyanate fitc anti epcam, by Thermo Fisher Scientific (3 mentions)
Mouse anti nanog, by BioLegend (3 mentions)
Mouse anti mmp 2, by Abcam (3 mentions)
Rabbit anti timp 1, by Abcam (3 mentions)
Mouse anti mmp 9, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fluorescently tagged secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gapdh, by Abcam (2 mentions)
Rat anti ly6g, by BioLegend (2 mentions)
33 protocols using rabbit anti cleaved caspase 3
1
Imaging Developmental Signaling Pathways
2
Western Blot Analysis of Apoptotic Markers
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis （SDS-PAGE） was
used to isolate the equal amounts of protein and transfer it to
polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The
membranes were blocked with 5% skim milk at room temperature, but
ser404p-tau protein membrane was blocked with 5%
bovine serum albumin (BSA). Then, incubate with primary antibody
overnight at 4°C (rabbit anti CD95, 1:300, Santa Cruz Biotechnology,
Santa Cruz, CA, USA; rabbit anti-CD95L, 1:400, rabbit anti-cleaved
caspase-3, 1:1000, rabbit anti-total tau, 1:1000, rabbit
anti-ser404p-tau, 1:1000, Abcam, Cambridge, MA, USA).
Then, the membranes were incubated with anti-rabbit secondary antibody
(1:500, Proteintech, Chicago, IL, USA) at room temperature for 1 h.
Finally, the bands were detected using enhanced chemiluminescence
(Beyotime, Shanghai, China). Image J (National Institutes of Health,
Bethesda, MD, USA) was used for quantitative band intensity;
glyceraldehyde 3-phosphate dehydrogenase (GAPDH，1:500, Abcam,
Cambridge, MA, USA) was used as the sample control.
used to isolate the equal amounts of protein and transfer it to
polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The
membranes were blocked with 5% skim milk at room temperature, but
ser404p-tau protein membrane was blocked with 5%
bovine serum albumin (BSA). Then, incubate with primary antibody
overnight at 4°C (rabbit anti CD95, 1:300, Santa Cruz Biotechnology,
Santa Cruz, CA, USA; rabbit anti-CD95L, 1:400, rabbit anti-cleaved
caspase-3, 1:1000, rabbit anti-total tau, 1:1000, rabbit
anti-ser404p-tau, 1:1000, Abcam, Cambridge, MA, USA).
Then, the membranes were incubated with anti-rabbit secondary antibody
(1:500, Proteintech, Chicago, IL, USA) at room temperature for 1 h.
Finally, the bands were detected using enhanced chemiluminescence
(Beyotime, Shanghai, China). Image J (National Institutes of Health,
Bethesda, MD, USA) was used for quantitative band intensity;
glyceraldehyde 3-phosphate dehydrogenase (GAPDH，1:500, Abcam,
Cambridge, MA, USA) was used as the sample control.
3
Bovine Mammary Cell Culture
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DMEM/F-12 and FBS were purchased from BI (FBS, BI, Kibbutz, Israel). Insulin, hydrocortisone, and collagenase I were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin was purchased from Gibco (Gibco Lab., Grand Island, NY, USA), and ovine prolactin was purchased from SGMEC (Israel). Rabbit anti-keratin 18 (CK18), rabbit anti-progesterone receptor (PR), rabbit anti-estrogen receptor α (ERα), rabbit anti-prolactin receptor (PRLR), rabbit anti-β-casein (CSN2), and rabbit anti-cleaved caspase-3 were obtained from Abcam (Cambridge, UK).
4
Podocyte Apoptosis in LPS Exposure
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Differentiated podocytes were exposed to LPS (100 ng/ml Escherichia coli 0111:B4 LPS (Sigma-Aldrich)) for 48 h. The activity of 100 ng/ml LPS in cell culture media was measured as described,1 (link) and was shown to be 1.7 EU/ml. When indicated, GIT27 (Tocris Bioscience) (10 μg/ml) was added to the cells 2 h before LPS exposure. Apoptosis was detected by FACS using Annexin V-FITC Kit and double staining with 7-AAD (BD, Franklin Lakes, NJ, USA) using FACSAria (BD). Cells positive for Annexin V-FITC and negative for 7-AAD were deemed apoptotic. A total of 1 × 104 cells were detected by FACS in each sample. Apoptosis was also detected by immunoblotting for total or cleaved caspase-3 as described above.
For In-Cell Western, podocytes were cultured in black 96-well plates (Perkin-Elmer, Waltham, MA, USA), fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS and permeabilized with 0.1% Triton X-100 in PBS. Cells were incubated with rabbit anti-cleaved caspase-3 (Abcam) at room temperature for 1 h, followed by IRDye 800 (LI-COR) donkey anti-rabbit IgG and 1 μM DRAQ5 (Thermo Fisher Scientific, Waltham, MA, USA) incubation at room temperature for 1 h. Detection and quantification was performed with an Odyssey Infrared Imager (LI-COR). The signal for cleaved caspase-3 was normalized with DRAQ5.
For In-Cell Western, podocytes were cultured in black 96-well plates (Perkin-Elmer, Waltham, MA, USA), fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS and permeabilized with 0.1% Triton X-100 in PBS. Cells were incubated with rabbit anti-cleaved caspase-3 (Abcam) at room temperature for 1 h, followed by IRDye 800 (LI-COR) donkey anti-rabbit IgG and 1 μM DRAQ5 (Thermo Fisher Scientific, Waltham, MA, USA) incubation at room temperature for 1 h. Detection and quantification was performed with an Odyssey Infrared Imager (LI-COR). The signal for cleaved caspase-3 was normalized with DRAQ5.
5
Immunocytochemical Analysis of Neural Markers
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Cells were fixed with 4% paraformaldehyde, and rinsed twice with 1× PBS. Primary antibodies included rabbit CD86 (1 μl/ml; Abcam, Cambridge, MA), rat CD206 (5 μl/ml; Abcam), rabbit Beta III Tublin (5 μl/ml, Abcam), rabbit anti-cleaved caspase-3 (0.5 μl/ml; Abcam), and mouse NeuN (10 μl/ml; Millipore, Billerica, MA). In addition, cell nuclei were stained with DAPI (0.5 μl/ml; Life Technologies, Carlsbad, CA). Fluorescently tagged secondary antibodies (Invitrogen) were visualized with an Olympus BX43 fluorescent microscope with a CellSens Standard imaging program. Five randomly selected images were taken per sample and each image was qualitatively assessed for overall fluorescence. NeuN-positive cells were manually counted using NIH ImageJ (Bethesda, MD).
6
Immunofluorescence Staining of Muscle Cryosections
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Muscle cryostat sections 8 μm-thick were incubated with blocking solution (0,25% Triton X-100, 3% Albumine from Bovine Serum in PBS) for 1h at RT. Subsequently, cells were incubated for 2 h at RT in a hydration chamber with the following primary antibodies at 1:50 dilution: rabbit anti-Aβ AB5078P (Millipore), rabbit anti-Bax and rabbit anti-cleaved Caspase3 (Abcam). Then, cells were washed thrice with PBS and incubated with Alexa Fluor 488 or 555 goat anti-mouse antibodies 1:500 for 2 h at RT. After 3 PBS washes, cells were incubated with 1μM Topro-3 for 10 min, washed with PBS and mounted with Mowiol. Digital images were taken at RT with a Leica TCS SP confocal microscope with ×40 objective and analyzed with Leica confocal software.
7
Immunohistochemical Staining of Skin Cryosections
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Skin punches were fixed with 4% formaldehyde, overnight at RT. The punches were washed in PBS, embedded in tissue freezing medium (Leica Biosystems), snap-frozen in a dry ice-ethanol bath and stored at –80°C. Leica cryostat CM1950 was used to cut 8 μm-thick cryosections that were mounted onto SuperFrost Plus adhesion slides (Thermo Fisher Scientific). Cryosections were permeabilized with staining buffer (0.3% Triton-100, 3% BSA in PBS) for 1h at RT, in a humidity chamber, and stained with antibodies. The following antibodies were used: rabbit anti-wide spectrum cytokeratin (Abcam), rabbit anti-cleaved caspase-3 (Abcam), anti-Staphylococcus aureus antibody biotin (Abcam), Alexa Fluor 647 Phalloidin (Life technologies), goat anti-rabbit Alexa Fluor 568 (Life Technologies) and streptavidin Alexa Fluor 488 (Life technologies). Primary antibodies were incubated for 1h at RT and, after three washes with PBS, secondary antibodies were added for 30 min at RT, in the dark. After antibody staining, slides were mounted with ProLong Gold Antifade Reagent with DAPI (Life Technologies). Confocal images were acquired using Zeiss LSM 700 confocal microscope.
8
Antibody-based DNA Damage Detection
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The following antibodies were used: mouse anti-yH2AX (Millipore), mouse anti beta-actin (Abcam) and IRDye 680RD donkey anti-mouse (LI-COR), rabbit anti-cleaved caspase 3 (Abcam), mouse anti y-H2AX (Millipore), anti-rabbit/555 (Invitrogen) and anti-mouse/488 (Life technologies). Chemicals were purchased from Sigma-Aldrich. MTH1 inhibitors were synthesized in-house and dissolved in DMSO to 10 mM [10 (link), 12 (link)].
9
Regulation of LRH-1 Signaling Pathway
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LRH-1 agonist DLPC (R-2, 3-bis dodecanoyloxy propyl 2-trimethylammonio ethyl phosphate, Cat#: B7661) was purchased from ApexBio (Boston, MA, USA). Rabbit anti-LRH-1 (Cat#: OM108702), rabbit anti-β actin (Cat#: OM241350) polyclonal antibodies were purchased from Omnimabs (Alhambra, CA, USA). Rabbit anti-progesterone receptor (Cat#: 49338) monoclonal antibody was purchased from Signalway antibody LLC (Greenbelt, MD, USA). Rabbit anti-Bcl2 (Cat#: 12789-1-AP) polyclonal antibody was purchased from Proteintech Group, Inc. (Wuhan, China). Rabbit anti-cleaved-caspase 3 (Cat#: ab49822) polyclonal antibody was purchased from Abcam (Cambridge, UK). Unless otherwise indicated, the other reagents and chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).
10
Hippocampal Protein Analysis Using Western Blot
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Hippocampus tissues were removed and homogenized in neuronal Protein Extraction Reagent (Thermo Fisher Scientific, 87792) containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, 87786). Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, 23227). Equal amounts of total protein were separated in 12% SDS–PAGE gels and then transferred to nitrocellulose (NC) membranes (Solarbio, Beijing, China). The primary antibodies were: rabbit anti-ADH1B (Biorbyt, 1:800), rabbit anti-BACE 1 (Abcam, 1:1000), rabbit anti-IDE (Abcam, 1:1000), rabbit anti-p75NTR (Cell Signaling Technology, 1:1000), rabbit anti-cleaved caspase-3 (Abcam, 1:1000), rabbit anti-Bcl-2 (Abcam, 1:1000), and rabbit anti-Bax (Abcam, 1:1000). Image Lab (Bio-Rad) was utilized for protein signal densitometry. Detection of proteins from pretreated SH-SY5Y cells using western blotting were performed as previously described (Zhang et al., 2016 (link)).
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