Apoptag tunel kit

The TUNEL experiment was performed using ApopTag TUNEL Kit (Millipore, S7165) to stain apoptotic cells following the manufacturer’s instructions. Briefly, animals were treated with 5% NAC (diluted in PBS) to remove the mucus for 5 min with gentle agitation at room temperature, followed by fixation with 4% formaldehyde (0.3% TritonX-100) for 30 min at room temperature. After bleached in 6% H₂O₂ (diluted in PBST) in bright light overnight, the animals were incubated with a TdT reaction mixture for 4 h at 37 °C. Next, the samples were washed and blocked before the fluorescent-labeled anti-DIG antibody (1:1000, Millipore, S7165) was added and incubated for 4 h at room temperature. After extensive wash for 4 h in PBSTx, samples were mounted and images were obtained with Leica SP8 confocal microscope. ImageJ was used for the quantification of histological images.

Rabbit polyclonal IgG anti-bovine serum albumin was purchased from MP Biochemical. Alexa fluor 488 conjugated bovine serum albumin was obtained from Invitrogen. Anti-β-actin, anti-pERK and anti-p38 MAPK antibodies were purchased from SantaCruz Biotechnology. Anti-GABA_BR2, anti-pCREB, and anti-active caspase-3 antibodies were obtained from Cell Signaling Incorporated. CGP52432 was obtained from Abcam (Cambridge, MA, USA). Anti-GABA_BR2 antibody was obtained from Thermo Scientific (Rockford, IL). Hematoxylin and eosin was obtained from Fischer Scientific (Pittsburgh, PA, USA). Anti-GAPDH and anti-TNF-α, and the ApopTag (TUNEL kit) were obtained from Millipore. All other reagents utilized in this study were obtained from Sigma Aldrich (St Louis, MO, USA) unless otherwise stated. Naphthol AS-D chloroacetate esterase (NACE) kit was obtained from Sigma Aldridge.

The TUNEL experiment was performed with ApopTag TUNEL Kit (Millipore, S7165) to determine apoptotic cell numbers in planarians following the manufacturer's instructions. Five per cent NAC (diluted in PBS) was used to remove the mucus coat of planarians. Planarians were fixed with 4% formaldehyde (with PBST containing 0.3% TritonX‐100) for 30 min at room temperature. The samples were then bleached in 6% H₂O₂ (diluted in PBST) in direct light overnight. The animals were incubated with TdT reaction mixture for 4 h at 37°C. The antibody was incubated for 4 h at room temperature. Animals were washed every half an hour for 4 h in PBST to reduce nonspecific labelling. Images were obtained with Leica SP8 confocal microscope. ImageJ was used for quantifications.