Anti caspase 8

Biopsies were obtained from 180 patients undergoing surgical resection of CRC at the Cancer Referral Center of Natal, Brazil. Additional clinical information has been previously described together with the method to generate the tissue microarray (TMA) blocks [37 (link)]. This research was approved by the institutional committee (No. 030/0030/2006, 20th July, 2006).
For immunohistochemistry (IHC), anti-BCL-2, anti-caspase-8, and anti-Ki-67 antibody were used (Abcam, Burlingame, CA, USA) at a dilution of 1:1500. The number of positive cells within each TMA core was counted under a light microscope with a high-power magnification (40×).
BCL-2, caspase-8, and Ki-67 expression in the tumor tissue and surrounding stromal tissue was independently assessed by two researchers, who were blinded to the clinical data. Disease staging was performed according to the modified Dukes’ criteria classification [37 (link)].

Lung cancer cells were lysed in a lysis buffer and protein samples were run on 8% SDS polyacrylamide gel and then transferred to Nitrocellulose (NC) membrane. After incubated with the specified primary antibody, and then corresponding horseradish peroxidase (HRP) conjugated secondary antibody, specific protein was developed by enhanced chemiluminescence (ECL) luminescence reagents (Amersham, UK). β-actin was applied as reference control. All antibodies (anti-PI3 kinase p85 alpha, anti-caspase 8, anti-MyD88 and anti-BIRC3 antibodies) were purchased from Abcam (Eugene, USA).

Protein samples were prepared as described above and subjected to western blot analysis. The samples (50 μg) were separated by SDS-PAGE (12.5% or 15% gel) at 100 V for 2 h. Gels were then transferred to a NC membrane that had been presoaked for 10 s in transfer buffer (25 mM Tris, pH 7.4, 192 mM glycine, and 20% methanol) at 300 mA for 1.5 h or 2 h. Each NC membrane was blocked for 2 h with 0.5% dried skim milk in TBS-T (20 mM Tris, 500 mM NaCl, and 0.05% v/v Tween 20) at RT, washed three times for 15 min each with TBS-T, and then incubated with a specific primary antibody (anti-TNF-α, anti-TNFR1, anti-active caspase-3, anti-caspase 8, and anti-GAPDH, Abcam, USA) in TBS-T with gentle shaking overnight at 4°C. Each membrane was washed three times for 15 min each with TBS-T and then incubated with the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG) in TBS-T with gentle shaking at 37°C for 1 h. Each membrane was rinsed three times for 15 min each with TBS-T, developed with the Super Enhanced Chemiluminescence Detection kit (GE healthcare, USA). All the membranes were exposed in and scanned by a ChemiDoc XRS+ (Bio-Rad). A semiquantitative analysis based on the OD values was performed by Image Lab Software (Bio-Rad), using ANOVA to compare groups.