Anti caspase 3 antibody

DNA fragmentation during apoptosis was detected using a TUNEL assay with the In Situ Cell Death Detection Kit (#12156792910; Roche, Indianapolis, IN). Paraffin-embedded sections were cleared with xylene and rehydrated according to the manufacturer’s instructions. After three washes in PBS, sections were preincubated inside a dark humidified chamber with 50 μL of TUNEL reaction mixture (5 μL of TUNEL-Enzyme solution + 45 μL of TUNEL-Label solution; tetramethylrhodamine-labeled dUTP) for 60 min at 37 °C and then counterstained with DAPI (#D9542; Sigma-Aldrich). The sections were mounted with mounting medium after washing three times with PBS. Immunofluorescence staining of active caspase-3 was performed with anti-caspase-3 antibody (#269518; R&D Systems) and Alexa Fluor 488-conjugated donkey anti-goat IgG (#A11055; Invitrogen), followed by counterstaining with DAPI. The sections were mounted with mounting medium after washing three times with PBS. Slides were imaged using the LSM 880. Quantification was performed using ZEN software.

Apicularen A was provided by Dr. Ahn (Division of Ocean Science, Korea Maritime University, Busan, Korea) and dissolved in dimethyl sulfoxide. Phorbol 12-myristate 13-acetate (PMA), thiazolyl blue tetrazolium bromide (MTT), anti-α-tubulin and anti-β-tubulin antibodies were purchased from Sigma (St Louis, MO, USA). Anti-PARP and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caspase-3 antibody was purchased from R&D Systems (Wiesbaden, Germany). Z-VAD-fmk, Ro31-8220 and Go6983 were purchased from Calbiochem (San Diego, CA, USA). All other reagents were molecular biology grade.