Pfkfb4 antibody

The breast cancer tissue microarrays (TMAs) were obtained from Roswell Park Comprehensive Cancer Center according to the Institutional Research Board approved protocol BDR099518. Clinical Data Delivery and Honest Broker services for this study were provided by Roswell Park Biomedical Data Science Shared Resource. PFKFB4 Antibody (Abcam, Cat. No. ab137785) at 1/100 was used for staining. Quantification of cytoplasmic and nuclear PFKFB4 staining was conducted by Roswell Park Pathology Network Shared Resource. See method details for additional information about immunohistochemical staining and quantification.

Formalin fixed paraffin sections were cut at 4 μm, placed on charged slides, and dried at 60°C for one hour. Slides were cooled to room temperature (25°C) and added to the Leica Bond Rx, where they were deparaffinized with Bond Dewax Solution (Leica, Cat. No. AR9222) and rinsed in water. Bond Epitope Retrieval 2(Leica, Cat. No. AR9640) was used for target retrieval for 30 min. Slides were blocked using peroxide block from Bond Polymer Refine Detection kit (Leica, Cat. No. DS9800) for 5 min. Slides were incubated with PFKFB4 Antibody (Abcam, Cat. No. ab137785) at 1/100 for 20 min followed by Rabbit Envision (Agilent, Cat. No. K4003) for 30 min. DAB (Diaminobenzidine) from the Bond Polymer Refine Detection kit (Leica, Cat. No. DS9800) was applied for 10 min for visualization. Slides were counterstained with Hematoxylin from the Bond Polymer Refine Detection kit (Leica, Cat. No. DS9800) for 8 min then placed into water. After removing slides from the Bond they were dehydrated, cleared, and coverslipped. Biospecimens or research pathology services for this study were provided by the Pathology Network Shared Resource.

Cells were seeded on a coverslip in 24-well tissue culture plate and incubated in regular cell culture incubator or hypoxia incubator for 24 h. Cells were first fixed with 10% buffered formalin for 10 min at room temperature (25°C) and then permeabilized with PBST (PBS with 0.25% Triton X-100) for 30 min at room temperature (25°C). Cells were blocked with 2.5% goat serum (Vector Labs, Cat. No. S-1012), followed by overnight incubation with PFKFB4 antibody (1:300, Abcam, Cat. No. 137785). Cells were then incubated with fluorochrome-conjugated secondary antibody (1:750, Thermofisher) and mounted in Anti-fade Mounting Medium with DAPI (Vector Laboratories, Cat. No. H-1800).