The tumor tissues from mice were washed with 1 × PBS. then fixed with 10% neutral buffered formalin, and embedded in paraffin using standard protocols. Then, tissue slices were deparaffinized in xylene and rehydrated in a graded series of ethanol. Slides were soaked to block endogenous peroxidase with 3% H2O2. For antigen retrieval, tissue slices were heated in 95–100 °C Tris-EDTA Buffer (101 mmol/L Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH9.0) for 30 min. The sectioned slides were incubated with a diluted primary antibody, Ac-Histone H3 (BD Bioscience San Jose, CA) or cleavage caspase 3 (Cell Signaling, Beverly, MA), or stained with hematoxylin and eosin. After rinsing with PBS several times, the secondary antibody, HRP polymer conjugate reagent A was applied to the slices, and subsequently reagent B for peroxidase catalyzation, which distinguishes the site of antigen. In each incubation step, the slides were followed by washing with PBS for 5 min. Then, Mayer’s Hematoxylin solution was used for counterstaining. The color of nuclei of cells was blue, and cytoplasm was pink. It could help to distinguish the nuclei and cytoplasm of tumor cells. The quantifications of Ac-Histone H3 and cleavage caspase 3 expression were analyzed by HistoQuest software (TissueGnotics GmbH, Vienna, Austria).
Cleavage caspase 3
Manufactured by Cell Signaling Technology
Sourced in United States
Cleavage caspase 3 is a lab equipment product that detects the cleaved form of caspase-3, a key executioner of apoptosis. It is used to monitor the activation of caspase-3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Cleavage parp, by Cell Signaling Technology (2 mentions)
Aurora a, by Cell Signaling Technology (2 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Phospho dna pkcs, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
Image pro plus version 7, by Media Cybernetics (1 mentions)
Mounting medium, by Merck Group (1 mentions)
710 laser scanning confocal microscope, by Zeiss (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl detection reagent, by Cytiva (1 mentions)
P chk1, by Santa Cruz Biotechnology (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
P atr, by Cell Signaling Technology (1 mentions)
P chk2, by Santa Cruz Biotechnology (1 mentions)
10 protocols using cleavage caspase 3
1
Immunohistochemical Analysis of Tumor Tissues
2
Evaluating Tumor Cell Biomarkers in Mice
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Mice from each group were sacrificed on day 21. The tumor was fixed in 10% neutral buffered formalin and processed for histopathological and immunohistochemical staining. After fixation, tumor tissues were embedded in paraffin blocks and sectioned (5 µm). Tumor cells were detected in representative stained sections. The expression of phospho-DNA-PKcs (#74965), phospho-Akt (#4060), cleavage caspase 3 (#9964; Cell Signaling Technology, Beverly, MA, USA), and MICA/B (ab203679; Abcam, Cambridge, UK) were evaluated after immunohistochemical staining with specific antibodies. Pictures were recorded using a Zeiss AxioImager microscope. Images of immunohistochemical staining with MICA/B were analyzed in an unsupervised and blinded fashion using TMARKER, a software toolkit for histopathologic staining estimation.21 (link) Immunoreactivity was analyzed by three investigators.
3
Immunofluorescent Labeling of Flavivirus Antigens
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The cultured cells were fixed in 4% paraformaldehyde for 20 min at room temperature and then incubated with methanol for 20 min at −20 °C. Fixed cells were blocked with 3% bovine serum albumin in PBS and then incubated with primary antibodies to Flavivirus Group Antigen (clone D1-4G2-4-15, Millipore, Billerica, MA), GFAP (Dako), or cleavage caspase 3 (Cell signaling Technology) overnight. At the second day, the cells were washed with PBS for three times and incubated for 1 h at room temperature with the secondary anti-mouse IgG antibody (coupled with green dye, Alexa Flour 488, Molecular Probes, Eugene, Oregon). Nuclear DNA were labeled with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO) for 10 min after the secondary antibody at room temperature. Cover slips were mounted on glass slides with mounting medium (Sigma-Aldrich). Fluorescent images were obtained using a Zeiss 710 Confocal Laser Scanning Microscope (Carl Zeiss, Oberkochen, Germany). All obtained images were imported into Image-ProPlus, version 7.0 (Media Cybernetics, Sliver Spring, MD) to quantify the number of infected cells. The assessors were blinded during image acquisition or quantification.
4
Western Blot Analysis of DNA Damage Response
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Protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, UK). Blots were blocked for 1 h in 5% milk/0.1% Tween 20 in phosphate buffered saline (PBS-T) and then incubated with primary antibodies (1: 1000) at 4°C overnight. Blots were then washed three times for 15 min in PBS-T, followed by incubation with secondary antibody (according to different primary antibodies, HRP-conjugated goat anti-mouse, anti-rabbit, and rabbit anti-goat IgG were used (1: 5000, Santa Cruz, Dallas, TX)) in 5% milk/PBS-T for 1 h, and then washed three times for 15 min in PBS-T. The membranes were briefly incubated with ECL detection reagent (Amersham Biosciences, Castle Hill, Australia) to visualize the proteins and were then exposed on X-ray film. Primary antibodies used were as follows: ATM (Cat# 600-401-398), and p-ATM (Cat# 600-401-400) were purchased from Rockland Immunochemical (Gilbertsville, PA, USA); cleavage-caspase-3 (Cat# 9661), cleavage-PARP (Cat# 5625P), γ-H2Ax (Cat# 9718), Bax (Cat# 2772s), and Bcl-2 (Cat# 2870), ATR (Cat# 2790), p-ATR (Cat# 2853S) were from Cell Signaling Technology (Beverly, MA); PARP (Cat# 7150), pro-caspase-3 (Cat# 7272), β-Actin (Cat# 1615), Chk1 (Cat# 377231), p-Chk1 (Cat# 2341), Chk2 (Cat# 8813), p-Chk2 (Cat# 2661) were from Santa Cruz (Dallas, Texas, US).
5
Comprehensive Protein Expression Analysis
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Western blot were performed as previously described22 (link),31 (link). The antibodies used were as follows: Aurora-A (cell signaling, #91590), Phospho-Aurora-A (Thr288) (cell signaling, #3079), mTOR (cell signaling, #2983), Phospho-mTOR (Ser2448) (cell signaling, #5536), AKT (cell signaling, #4691), Phospho-AKT(Ser473) (cell signaling, #4060), ERK (cell signaling, #4695), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (cell signaling, #4370), FLAG (Sigma-Aldrich, # F4042;), E-cadherin (cell signaling, #14472), p21 (cell signaling, #2947), ki-67 (cell signaling, #9449), PARP (cell signaling, #9532), cleavage Caspase-3 (cell signaling, #9661), and β-actin (cell signaling, #14793).
6
Western Blot Analysis of Fibrosis Markers
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Thirty micrograms of protein were dissolved in SDS-PAGE gel, then transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich) by electroblotting. After electroblotting, the membranes were blocked for 1 hour at room temperature with 5% blocking solution, the required bands were cut according to marker, and THBS1 antibodies (1:1000, Cell Signaling Technology, Cat# 37879), TGF-β1 (1:1000, Abcam, ab92486), α-SMA (1:500, Abcam, ab5694), Collagen I (Col-1, 1:1000, Abcam, ab6308), Cleavage Caspase 3 (1:1000, Cell Signaling Technology, Cat# 9661), and β-Actin (1:1000, Abcam, ab8227) were incubated overnight at 4°C. The second antibody (1:5000) was incubated at room temperature for 1 hour. Immunoreactivity was measured using the Western Lighting Ultra Kit (ECL, Pierce Technology, Shanghai, China).
7
Protein Expression Analysis in Lung Cancer
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NCI-H23 and A549 cells were lysed with RIPA lysis solution (Beyotime) containing a mixture of protease inhibitors. The cell protein lysis product was separated by 10% SDS-PAGE electrophoresis, transferred onto a 0.22 mm PVDF membrane (Millipore), and detected with a specific antibody. A chemiluminescent substrate was added to the specific strip and quantified with densitometry (Quantity One software, BioRad). The GAPDH antibody was used as a control. Anti-Caspase-3 antibody, cleavage Caspase-3, poly (ADP-ribose) polymerase, cleavage PARP, BAX, BAK, cyclin D3 and CDK4 (1:1000) were purchased from Cell Signaling Technology. Anti-SIRT6 antibody was purchased from Abcam. BCL-2 and BCL-XL antibody were purchased from Proteintech.
8
Western Blot Analysis of Cell Signaling
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For direct IB analysis, cells were lysed in a Triton X-100 or RIPA buffer with phosphatase inhibitors. The antibodies used were as follows: HA (1:2000; Roche), CycG1 (1:500; Santa Cruz), PARP (1:1000; Cell Signaling), Cleavage-caspase-3 (1:500; Cell Signaling), Aurora A (1:1000; Cell Signaling), NOXA (1:1000; Millipore), Mcl-1 (1:1000; Cell Signaling), Bcl-2 (1:1000; Cell Signaling), Bcl-xL (1:1000; Cell Signaling), CycB1 (1:1000; Cell Signaling), p-Histone 3 (1:500; Cell Signaling) and β-actin (1:5000; Sigma).
9
Amyloid-beta Induced Apoptosis Signaling
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In a set of experiments, increasing concentrations (0.1, 0.3, 1 and 3 μm ) of Aβ42O were added for 24 h to the culture medium of SH‐SY5Y cells seeded on six‐well plates. In another set of experiments, SH‐SY5Y cells were plated on six‐well plates and then treated for 24 h with Aβ42O at 3 μm . Cells were then collected and lysed 30 min at 4 °C in a buffer containing 50 mm Tris, pH 7.5, 120 mm NaCl, 1 mm EDTA, 6 mm EGTA, 15 mm Na4P2O7, 20 mm NaF, 1% Nonidet and protease inhibitor cocktail. Then, lysates centrifuged at 10 000 g , 15 min at 4 °C and 15 μg of protein from total cell lysates were used to perform SDS/PAGE and western blotting analysis in order to evaluate with specific antibodies caspase 3 cleavage (Cell Signalling) or expression of SK1 (ECM Biosciences), SK2 (ECM Biosciences), Spns2 (kindly gifted by T. Nishi). PDVF membranes were incubated overnight with the primary antibodies at 4 °C and then with specific secondary antibodies for 1 h at room temperature. The binding of the antibodies with the specific proteins has been detected by chemiluminescence employing Amersham Imager 600 (GE Heathcare, Buckinghamshire, UK). Densitometric analysis was performed by imagej software.
10
Evaluating ALKAL1 Expression in Colorectal Cancer
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Immunohistochemical staining was performed on 4 μm tissue sections using an EnVision™ Kit (DAKO, Denmark). After deparaffinized, rehydrated in graded ethanol, antigen retrieval and blocking, the slides were incubated overnight at 4 °C in a humidified chamber with anti-ALKAL1 antibodies (Thermo Fisher, PA5-55591), Vimentin (sc-373717), Ki67 (27309-1-AP, Proteintech) and caspase 3 cleavage (Asp175, 9661, Cell Signaling Technology) diluted 1:50 in PBS. Scores given by two independent Pathologists were averaged for further comparative evaluation of ALKAL1 expression. The details of scoring criteria were described in our previous study 26 (link), 27 . According to this method, ALKAL1 expression in colorectal cancer samples was evaluated by the staining index, with scores of 0, 1, 2, 3, 4, 6, 8, 9 or 12. High or low expression of ALKAL1was stratified according to the following criteria: SI ≤ 4 was defined as tumors with low expression of ALKAL1, and SI ≥ 6 was defined as tumors with high expression of ALKAL1.
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