Tdt fragel dna fragmentation detection kit

Brains from control and MV-infected mice (7 dpi) were collected following the procedure for the immunofluorescence assay. Sagittal brain sections were fixed in 3.7 % formaldehyde and subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; (TdT-FragEL DNA fragmentation detection kit; Millipore) using diaminobenzidine as a substrate. For counting of TUNEL-positive cells, four non-overlapping fields (×20) were chosen by a blinded examiner moving dorsally to ventrally across the slice. TUNEL-positive cells were counted on an Olympus BX41 Laboratory Microscope (Olympus Corporation). Three sagittal slices were counted from each brain, with three mice assayed per condition. The average number of TUNEL-positive cells per ×20 field ± standard error was determined.

Apoptotic cell death in tumor xenograft tissue sections was determined by TUNEL assay using the TdT-FragEL DNA Fragmentation Detection Kit (EMD Millipore, Burlington, MA) as described previously [24 (link)]. Briefly, sections were digested with proteinase K, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide in 10 mM Tris (pH 8.0). The sections were then placed in equilibration buffer and incubated with TdT enzyme in a humid chamber at 37° C for 1.5 h. The apoptotic nuclei were stained by 3,3′-diaminobenzidine and observed by microscopy. We manually counted the number of positively stained nuclei and the percentage of positive cells versus the total number of cells was calculated.

5μm tumor sections were deparaffinized and rehydrated. TUNEL analysis was performed using the TdT-FragEL DNA Fragmentation Detection Kit (EMD Millipore, Billerica, MA) following the manufacturer's directions. At least 1000 cells and at least 3 sections per animal were analyzed.

Clivorine was isolated from L. hodgsonii Hook and identified by IR, NMR, and MS with 99.5% purity [4] (link). Quercetin was purchased from Sigma Chemical Co. (St. Louis, MO). Terminal dUTP nick end-labeling (TUNEL) staining system (TdT-FragEL DNA Fragmentation Detection Kit) was purchased from Merck Calbiochem (Darmstadt, Germany). The kits for determining serum alanine/aspartate transaminase (ALT/AST) activity, and total bilirubin (TB) level were obtained from the Shanghai Rongsheng Biotech Corporation (Shanghai, China). Anti-caspase-3 antibody was purchased from Cell Signaling Technology (Danver, MA). Peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (H+L) was purchased from Jackson ImmunoResearch (West Grove, PA). BCA Protein Assay Kit was purchased from Thermo Scientific (Rockford, IL). Anti-4 Hydroxynonenal (4-NHE) antibody was purchased from Abcam (Cambridge, UK). The DAKO EnVision detection system was purchased from DAKO Corporation (Carpinteria, CA). Trizol reagent was purchased from Life Technology (Carlsbad, CA). PrimeScript® RT Master Mix and SYBR Premix Ex Taq were purchased from Takara (Shiga, Japan). RT² Profiler PCR array was purchased from Qiagen (Hilden, German). All other reagents were purchased from Sigma (St. Louis, MO), unless otherwise indicated.

Paraffin-embedded tissues were cut into sections and TUNEL assays were performed using a TdT-FragEL™ DNA Fragmentation Detection Kit (QIA33, Merck) according to the manufacturer’s instructions. Sections were examined and photographed using a light microscope.

TUNEL assay was performed to detect the apoptotic cells after 3 days in culture using the TdT-FragEL DNA Fragmentation Detection Kit (QIA33, Merck, Darmstadt, Germany) according to the manufacturer’s instructions. Apoptosis of tissue samples was evaluated using another TUNEL apoptosis detection kit (DAB type, Wanlei Biotechnology). TUNEL-positive cells were counted, and the apoptotic rate was calculated as follows: positive cells / (positive cells + negative cells) × 100%.