Anti caspase 3 antibody

Sodium citrate buffer (0.01 M and pH 6) was used on testis sections with an antigen retrieval step. UltraVision detection system (Thermo-Scientific, USA) was applied in immunohistochemistry assays, as previously described by Urriola-Muñoz, P. et al.^{22 (link)}. Slides were incubated overnight at 4 °C with an anti-caspase 3 antibody (Cell Signaling, USA) at 1 mg/ml and the sections counter-stained with hematoxylin and subsequently evaluated under a microscope. Active caspase-3 cells were quantified in a minimum of 100 seminiferous tubules per each replicate, per (n).

All rats were sacrificed in the 10th week after treatment, and then necropsy was performed. The total weight of the bladder was then determined as previously described [25 (link)]. Tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. For morphological analysis, H&E staining and microscopic examination were performed on 3 µm-thick sections from paraffin-embedded tumor blocks. Immunohistochemical staining was probed with polyclonal anti-Caspase 3 antibody and polyclonal anti-E-cadherin antibody (Cell Signaling Technology, USA) according to the manufacturer’s instructions. Data was analyzed by Image-pro Plus Analysis system (Olympus, Japan). Macrophages were separated from tumor cells by their expression of CD11b and F4/80 for mouse macrophages and determined by flow cytometry [21 (link)].

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.

Cell extracts were diluted in sample buffer (50 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 6% 2-mercaptoethanol, and 0.0025% bromophenol blue) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were loaded at the same protein concentration for each experiment. The primary antibodies used were anti-LMP1 antibody (S12) at 1:50, anti-actin antibody (AC-74, Sigma, St. Louis, MO) at 1:5000, anti-phospho-AKT antibody (#4058, Cell Signaling Technology, Danvers, MA) at 1:1000, anti-AKT antibody (#9272, Cell Signaling Technology) at 1:1000, anti-NFκB (p65) antibody (610868, BD Biosciences, Franklin Lakes, NJ) at 1:250, anti-IκBα antibody (#4814, Cell Signaling Technology) at 1:1000, anti-caspase-3 antibody (#9662, Cell Signaling Technology) at 1:1000, and anti-poly(ADP-ribose) polymerase (PARP) antibody (C-2-10, Sigma) at 1:2000. The secondary antibodies used were Goat Anti-Mouse Ig's HRP Conjugate (AMI3404, BioSource International, Camarillo, CA) and HRP-Goat Anti-Rabbit IgG (H+L) (656120, Invitrogen, Carlsbad, CA). The bands were visualized using WEST-oneTM Western Blot Detection System (iNtRON Biotechnology, Seongnam, Korea) or Chemi-Lumi One Super (Nacalai tesque, Kyoto, Japan).

MDA-MB-231 cells were cultured in six-well plates (1.5 × 10⁵ cells/well) and incubated overnight. When the cells reached 70–80% confluence, the medium was aspirated and fresh DMEM containing 5-FU, PTX, 5-FU/PTX, 5-FU/PTX Lps, or KLA-5-FU/PTX Lps (5-FU: 3 µg/mL; PTX: 2 µg/mL) was added. After incubation at 37 °C for 12 h, the medium was aspirated, and the wells were washed three times with cold PBS. After incubation with a cell lysis reagent (Beyotime Biotechnology, Shanghai, China) for 5 min, the cell suspension was aspirated and centrifuged. Proteins in the supernatant were fractionated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and then incubated with anti-caspase 3 antibody (Cell Signaling Technology, Boston, MA, USA), as described elsewhere [34 (link)]. Antibody binding was measured by enhanced chemiluminescence, and the results were analyzed using ImageJ Software.