Anti cdk1

Formalin-immobilized tissues were dewaxed in xylene, rehydrated in ethanol solution, incubated with 3% hydrogen peroxide to block endogenous peroxidase and nonspecific binding sites. The tissues were incubated with the primary anti-ZNF655 (1:200, Invitrogen, PA5-56183), anti-CDK1 (1:50, Sigma, HPA003387) antibody at 4 °C overnight and then with the secondary antibody HRP goat anti-rabbit IgG (1:200, Beyotime, A0208) at room temperature for 1 h. After incubation with peroxidase conjugated streptavidin and diaminobenzidine, hematoxylin was stained. The staining intensity was scored according to the criteria described in the literature [23 ]. The result greater than or equal to median values of IHC was defined as ZNF655 high expression, otherwise low expression.

Tissue microarray (TMA) sections containing both normal and cancerous breast tissues were retrieved from Cooperative Human Tissue Network, the University of Virginia. TMAs were deparaffinized and rehydrated using standard methods. The sections were then sequentially incubated with rabbit polyclonal anti-CDK1 (Sigma, St. Louis, MO), biotin-conjugated goat anti-rabbit, and ExtrAvidin Peroxidase (ExtrAvidin Kit, Sigma). Staining was developed with liquid DAB substrate (DAKO, Carpinteria, CA), sections were counterstained with hematoxylin, and mounted with Permount (Fisher Scientific, Pittsburgh, PA). Stained TMAs were scanned, and digital images were obtained with Aperio Scanscope System (Leica Biosystems, Vista CA). The intensity of CDK1 staining in a tissue from each patient was evaluated with the Positive Pixel Count Algorithm (Leica Biosystems). This algorithm quantifies the amount of specific stain present in a digital slide by evaluating an average intensity of all pixels for subsequent calculation of the optical density and the proportion of positively stained area.