Proteinase k from tritirachium

For genotyping, the tip of the tail was cut and incubated in 50 µl Tris-acetate-ethylenediamine-tetraacetic acid buffer (Kanto Chemical Company Incorporated, Tokyo, Japan) at 95 °C for 5 min, then incubated in 1.5 µl Proteinase K from Tritirachium (Sigma-Aldrich, St. Louis, MO, USA) at 60 °C for 60 min and 100 °C for 5–10 min. Casp-3 KO mice were genotyped by standard polymerase chain reaction (PCR) using 1 µl tail lysate, Takara Ex Taq (Takara Bio, Shiga, Japan), 10X Ex Taq Buffer (Takara Bio), and dNTP Mixture (Takara Bio), according to the manufacturer’s instructions. Primers for Casp-3 KO mice (sequences: 5′-GCG AGT GAG AAT GTG CAT AAA TTC-3′, 5′-GGG AAA CCA ACA GTA GTC AGT CCT-3′, and 5′-TGC TAA AGC GCA TGC TCC AGA CTG-3′) were synthesized by Takara Bio and diluted in MilliQ water to 0.2 µM. For the PCR, a thermal cycler (Takara Bio) was programmed as follows: (1) 94 °C for 2 min; (2) 94 °C for 20 sec; (3) 65 °C for 15 sec, decrease by 0.5 °C per cycle; (4) 68 °C for 10 sec; (5) repeat steps 2–4 for 10 cycles; (6) 94 °C for 15 sec; (7) 60 °C for 15 sec; (8) 72 °C for 10 sec; (9) repeat steps 6–8 for 28 cycles; (10) 72 °C for 2 min; (11) 10 °C hold.