Xenograft tumors were dissected and fixed in 10% neutral-buffered formalin (Sigma-Aldrich) and subsequently embedded in paraffin. Five-micrometer-thick sections were prepared and stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed on formalin-fixed paraffin tumor sections, as previously described [47 (link)]. Primary antibodies used were anti-Ki-67 antibody (dilution 1:300; Thermo Scientific, Fremont, CA; #RB-9043-P0) and anti-cleaved caspase-3 antibodies (1:300 dilution; Cell Signaling, Cat #: 9661). Staining was developed with 3,30 diaminobenzidine (DAB) using the DAB substrate kit for peroxidase (Vector Laboratories, Burlingame, CA, SK-4100). For quantitative analysis Ki-67 or cleaved caspase-3 positive cells were counted by using NIH Image J software version 1.47 (Wayne Rasband, National Institutes of Health, Bethesda, MD).
Anti cleaved caspase 3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, Germany, Switzerland, China
The Anti-cleaved caspase-3 antibody is a laboratory tool used to detect the activated form of caspase-3, a key enzyme involved in the apoptosis (programmed cell death) pathway. The antibody specifically recognizes the cleaved form of caspase-3, which is a hallmark of cells undergoing apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Anti caspase 3 antibody, by Cell Signaling Technology (8 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Anti β actin antibody, by Cell Signaling Technology (6 mentions)
Anti bax antibody, by Cell Signaling Technology (5 mentions)
Anti ki67 antibody, by Cell Signaling Technology (5 mentions)
Vectastain abc kit, by Vector Laboratories (4 mentions)
Anti bcl 2 antibody, by Cell Signaling Technology (4 mentions)
Anti parp antibody, by Cell Signaling Technology (4 mentions)
Anti gapdh antibody, by Cell Signaling Technology (4 mentions)
Anti cox 4 antibody, by Cell Signaling Technology (3 mentions)
Anti β actin antibody, by Merck Group (3 mentions)
Rnase a, by Merck Group (3 mentions)
Anti cleaved parp antibody, by Cell Signaling Technology (3 mentions)
Anti actin antibody, by Merck Group (3 mentions)
In situ cell death detection kit, by Roche (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Neutral buffered formalin, by Merck Group (2 mentions)
Histofine simple stain max po, by Nichirei Biosciences (2 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
174 protocols using anti cleaved caspase 3 antibody
1
Xenograft Tumor Histological Analysis
2
Cancer Cell Proliferation and Apoptosis
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After 14 days of culture, cells were fixed with 10% formalin, routinely processed and embedded in paraffin. Deparaffinized sections were generated and histological observations were made after standard hematoxylin-eosin (HE) staining. Silver impregnation was used to evaluate the depth of cancer cell invasion. Proliferative cells were labeled with a mouse monoclonal anti-Ki-67 antibody (#M7240, Dako, Agilent Technologies, Santa Clara, CA, USA). Apoptosis of cells was detected using anti-cleaved caspase-3 antibodies (#9664; Cell Signaling Technology (CST), Danvers, MA, USA). Immunostaining for Ki-67 and cleaved caspase 3 was detected using Histofine® Simple Stain MAX PO (Nichirei, Tokyo, Japan). The percentages of Ki-67-positive cells and cleaved caspase-3-positive cells were determined as indicators of proliferation and apoptosis, respectively. The cancer cell layer thickness was measured in 10 areas in each of 5 randomly selected non-contiguous and non-overlapping areas (low magnification, ×10 objective). The depth of cancer cell invasion was measured from the basement membrane to the deepest region of the cancer cells.
3
Quantifying Apoptosis in Nerve Cells
4
Molecular Mechanisms of Temozolomide in Cancer
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KU0060648, Temozolomide, MK-2206, PD98059 were obtained from Merck Millipore. Anti-total DNA-PKcs, anti-phospho-DNA-PKcs (p-DNA-PKcs, Ser 2056) and anti-VEGF antibodies were purchased from Abcam. Anti-AKT, anti-p-AKT (Ser 473), anti-p-histone H2AX (γH2AX, Ser 139), anti-GSK3β, anti-p-GSK3β (Ser 9), anti-survivin, anti-Mcl-1, anti-c-Myc, anti-MMP9, anti-Ki-67, anti-cleaved caspase-3 antibodies, anti-Actin, anti-mouse and anti-rabbit secondary antibodies were purchased from Cell Signaling Technology.
5
Immunohistochemical Staining Protocols
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Pharmingen® rat anti mouse PECAM (CD31) monoclonal antibodies (Cat#550274) were purchased from BD Biosciences (Franklin Lakes, NJ, USA); mouse polyclonal anti α-SMA FITC-conjugated antibodies (Cat#F3777) were purchased from Sigma-Aldrich; rabbit polyclonal anti Ki-67 (Cat#ab16667) and rat monoclonal anti CD68 (Cat#ab5344) antibodies were purchased from Abcam (Cambridge, UK); rabbit polyclonal anti cleaved caspase-3 antibodies (Cat#9664S) were purchased from Cell Signaling Technology (Boston, MA, USA). Secondary fluorescent antibodies AlexaFluor®594-conjugated donkey anti rat (Cat#A21209) and AlexaFluor®594-conjugated donkey anti rabbit (Cat#A21207) were purchased from Invitrogen (Carlsbad, CA, USA);. Vectastain ABC kit (Rat IgG) (Cat#S-5000) was purchased from Vector Labs (Burlingame, CA, USA) and used for visualization of CD68 staining.
6
Immunocytochemical Analysis of Neural Cells
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Primary NSCs and neurons were plated on poly-D-lysine-coated six-well plate for 7 days. After conditioned medium treatment, the medium was decanted and the cells were washed three times with PBS; they were then fixed with 4% paraformaldehyde for 30 min. Then NSCs and neurons were permeabilized with 0.3% Triton X-100 in PBS for 15 min. After being blocked in 3% bovine serum albumin (BSA) with PBS at room temperature for 1 h, NSCs and neurons were washed again in PBS and finally incubated overnight at 4°C with one of anti-MAP 2, anti-nestin, or anti-cleaved caspase-3 antibodies (1 : 200, Cell Signaling Technology, USA). Cells were then washed with PBS and incubated in one of two secondary antibodies (TRITC-anti-rabbit (555 nm) and DyLight 488-anti-rabbit 1 : 400, Cell Signaling Technology, USA) for 2 h. A 10 min incubation in DAPI was used to counterstain nuclei. Finally, fluorescent images were captured on an epifluorescence microscope (Leica, Germany), and fluorescence intensity was measured with ImageJ software.
7
Protein Expression Analysis by Western Blot
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Cells were lysed using RIPA lysis buffer (Biotechnology, China) containing a protease inhibitor cocktail (Roche, Switzerland). Proteins were separated by 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA), incubated with 5% non-fat milk powder in TBST for 1 h at room temperature, and treated with specific primary antibodies overnight at 4°C. Then the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody, and each band was detected using an enhanced chemiluminescence kit (Millipore, MA, USA) and visualized with an enhanced chemiluminescence (ECL) BioImaging system (Azure, USA). Anti-CCBE1 was purchased from Biorbyt (catalog number orb215381). Anti-GAPDH, anti-caspase-3, and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (catalog numbers 2118S, 9663S, and 9661S, respectively). TUSC2 antibodies were purchased from Affinity Bioscience (catalog number AF0500). Anti-glutamate receptor 3/GRIA3[EP813Y] and anti-Bad were purchased from Abcam (catalog numbers ab40845 and ab40845, respectively). Anti-PDCD4 antibodies were purchased from Santa Cruz Biotechnology (catalog number sc-376430).
8
Cleaved Caspase-3 Staining in Kidney Tissue
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Left and right kidneys from WT and TRPC6 KO mice were harvested, fixed in 10% formalin for 24 h, embedded in paraffin, and then cut (5 µm) for cleaved caspase-3 staining. Sections were rehydrated, and antigens were unmasked in 10 mM of sodium citrate (pH 6.0) heated at 95°C for 30 min. Serum-free protein blocker (Vector Laboratories) was added, and sections were then incubated with anticleaved caspase-3 antibodies (No. 9661, Cell Signaling, dilution: 1:200) overnight at 4°C in a humid chamber. After being rinsed with PBS, sections were incubated with secondary antibody (1:400) and then subjected to diaminobenzidine staining by a Vector ABC-HRP Kit.
9
Western Blot Assay: Cell Lysis Protocol
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Total cell lysates were prepared in RIPA buffer (Cell Signaling) with complete protease inhibitor cocktail (Roche), phosphatase inhibitors (Roche), 5 mM dithiothreitol (DTT, Sigma) and 1 mM PMSF (Sigma) for 30 min on ice, and then centrifuged at 12,000 × g for 10 minutes at 4°C. The supernatant was recovered and protein concentration was determined by the Bio-Rad protein assay kit (Hercules, CA) according to the manufacturer's instructions. Western blot assays were performed as previously described [13 (link)]. The primary antibodies were obtained as follows: anti-SORBS1, anti-GAPDH, and anti-β-actin antibody from Abmart; anti-Hsp90, anti-Snail, anti-Slug, anti-vimentin, anti-JNK, anti-p-JNK, anti-c-Jun, anti-p-c-Jun, anti-p53, anti-caspase3, and anti-cleaved-caspase3 antibodies and horseradish peroxidase (HRP) -conjugated secondary antibodies from Cell Signaling Technology; and anti-E-cadherin and anti-N-cadherin antibodies from BD Transduction Laboratories.
10
Protein Extraction and Immunoblotting Assay
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Cell lysis buffer (Minute™ Protein Extraction Kits, Invent, MN, USA) was applied for cell collection. BCA protein assay (KGP902, KeyGEN BioTECH, Jiangsu, China) was operated to quantify protein concentration. Identical quantities of protein (30 μg/lane) were exposed to 12% SDS-PAGE before transferring to polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, USA) membranes. Following being blocked in 5% fat-free dry milk, anti-cleaved caspase-3 antibodies (1 : 1000, Cell Signaling Tech, Danvers, MA, USA) or anti-KLF6 antibody (1 : 1000, ab241385, Abcam, Cambridge, MA, UK) was applied to the membranes for incubation at 4°C overnight. Anti-GAPDH (1 : 5000, AP0066, Bioworld Tech, MN, USA) was used as an internal control. Following being washed, horseradish peroxidase-linked goat anti-rabbit IgG (1 : 3000, BS13278, Bioworld Tech, MN, USA) was applied to incubate the membranes for 1 h. Bound antibodies were identified with the implementation of the ECL detection system (Carestream, NY, USA), and the immunoreactive bands were quantified utilizing ImageJ 1.41o software (National Institute of Mental Health, USA).
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