Streptavidin biotin complex peroxidase kit

Water-soluble progesterone (PG), anesthetic MS-222, and an ATP Bioluminescence Assay Kit CLS II were purchased from Sigma (St. Louis, MO, USA). Human chorionic gonadotropin (hCG) was obtained from Teikoku Zoki (Tokyo, Japan), and collagenase (280 U/mg) was obtained from Wako (Osaka, Japan). Fluorogenic caspase-3 substrate IV was purchased from Calbiochem (La Jolla, CA, USA), and Apo-ONE homogenous caspase 3/7 assay was obtained from Promega (Madison, WI, USA). Polyclonal anti-cyclin B2 antibody was ordered from Santa Cruz (Dallas, TX, USA), and biotinylated anti-rabbit IgG was purchased from Vector Laboratories (Burlingame, CA, USA). Polyclonal anti-MAPK and anti-pMAPK antibodies were purchased from Cell Signaling (Beverly, MA, USA). The Streptavidin Biotin Complex Peroxidase Kit and CBB protein assay kit were obtained from Nacalai Tesque (Kyoto, Japan), and the bioluminescent ApoSENSOR ADP/ATP ratio assay kit was purchased from BioVision (Mountain View, CA, USA). Other chemicals were obtained from Wako and Nacalai Tesque.

Tissue blocks were prepared from formaldehyde-treated tissue using standard methods. Slides with 4 μm sections were heated at 60 °C for 15 min and then standard deparaffinization with xylene was performed. Antigen retrieval was carried out using an electric kettle at 98 °C for 40 min in 0.2 M citrate buffer at pH 6.0. Inactivation of endogenous peroxidase activity was performed in 0.3% H₂O₂/methanol at room temperature for 20 min. Sections were blocked with donkey serum for rabbit antibodies at room temperature for 30 min. The anti-ASCT2 (SLC1A5) antibody (#8057: CST Danvers, Danvers, MA, USA) was diluted 1/2000 0.1% BSA/PBS and Rabbit isotype control (#3900: CST Danvers, Danvers, MA, USA) was used at the same concentration. Sections were incubated with primary antibody overnight at 4 °C. Slides were washed three times in PBS and exposed to biotinylated anti-rabbit antibody (VECTORSTAIN Elite ABC HRP kit: PK-6101:VECTOR Laboratories, Burlingame, CA, USA) at room temperature for 30 min, then to the streptavidin-HRP at room temperature for an additional 30 min (Streptavidin-Biotin Complex Peroxidase kit: 30462-30: nakalai tesque, Kyoto, Japan). Color development utilized DAB (Peroxidase Stain DAB kit: 25985-50: nakalai tesque, Kyoto, Japan) and hematoxylin counterstaining was carried out in the standard fashion at room temperature for 5 min.