Avidin biotin complex detection kit

2HF (purity ~99%), MTT, Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against KRAS, pERK (T^202/204), pSTAT3 (Y⁷⁰⁵), CD31, Ki67, cyclin B1, CDK4, Bcl2, survivin, Bim, Bax, vimentin, fibronectin, and E-cadherin were purchased from Santa Cruz Biotechnology (Columbus, OH) and Cell Signaling Technologies (Danvers, MA). CellTiter-Glo was procured from Promega (Madison, WI). The avidin/biotin complex detection kit was procured from Vector (Burlingame, CA). The universal Mycoplasma detection kit was purchased from American Type Culture Collection (ATCC; Manassas, VA). The RLIP antibodies and antisense were obtained as previously described [19 (link),23 (link)]. D-luciferin was purchased from Goldbio (St. Louis, MO). AlamarBlue® was purchased from Thermo Fisher Scientific (Hanover Park, IL). The cell invasion assay kit was purchased from Cell Biolabs, Inc. (San Diego, CA).

Paraffin sections (3 μm) of formalin-fixed tissue were dewaxed and rehydrated. After heat-induced antigen retrieval, endogenous peroxidase activity and nonspecific reactions were blocked by 3% hydrogen peroxide in methanol and 1.5% normal goat serum, respectively. Sections were then incubated with anti-ITIH5 antibody (Sigma-Aldrich, St. Louis, MO) a dilution of 1:100 overnight at 4°C. The antigen and antibody complexes were visualized with an avidin biotin complex detection kit (Vector Laboratories, Inc. Burlingame, CA) and a diaminobenzidine substrate (Life Technologies, Grand Island, NY). The sections were counterstained with hematoxylin, and mounted. Normal placenta tissue, known to abundantly express ITIH5¹⁶, was used as positive control for ITIH5, and negative control was created by replacing the primary antibody with PBS. ITIH5 staining was considered as positive if at least 5% cancer cells were stained and was scored as follows: 0 (negative), 1 (weak), 2 (moderate), or 3 (strong).

2HF (purity ∼99%), DMBA, horseradish peroxidase (HRP)-conjugated anti-mouse, and anti-rabbit secondary antibodies, and MTT were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against pAKT (S⁴⁷³), PCNA, CD31, Ki67, CDK1, Bcl2, maspin, survivin, Bax, vimentin, and E-cadherin antibodies were purchased from Santa Cruz Biotechnology (Columbus, OH) and Cell Signaling Technologies (Danvers, MA). The avidin/biotin complex detection kit was procured from Vector (Burlingame, CA). Mayer’s hematoxylin, methyl salicylate, and Permount were purchased from Fisher Scientific (Hanover Park, IL). The source of RLIP antibodies was the same as previously described (16 (link)).