phospho-GSK-3 alpha (S21) Antibody

Detection of phospho-GSK3a in Human BreastCancer via seqIF™ staining on COMET™

Phospho-GSK3a was detected in immersion fixed paraffin-embedded sections of human breast cancer using Rabbit Anti-Human phospho-GSK3a Monoclonal Antibody (Catalog # AF4125) at 25ug/mL at 27 ° Celsius for 4 minutes. Before incubation with the primary antibody, tissue underwent an all-in-one dewaxing and antigen retrieval preprocessing using PreTreatment Module (PT Module) and Dewax and HIER Buffer H (pH 9; Epredia Catalog # TA-999-DHBH). Tissue was stained using the Alexa Fluor™ Plus 555 Goat anti-Rabbit IgG Secondary Antibody at 1:100 at 37 ° Celsius for 2 minutes. (Yellow; Lunaphore Catalog # DR555RB) and counterstained with DAPI (blue; Lunaphore Catalog # DR100). Specific staining was localized to the cytoplasm and nucleus. Protocol available in COMET™ Panel Builder.

Detection of Human and Mouse Phospho-GSK‑3 alpha (S21) by Western Blot.

Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with 200 nM PMA for 20 minutes and MCF-7 human breast cancer cell line untreated or treated with 100 ng/mL Recombinant Human IGF-1 (Catalog # 291-G1) for 15 minutes and NIH-3T3 mouse embryonic fibroblast cell line untreated or treated with 10 ng/mL PMA for 20 minutes. PVDF membrane was probed with 1 µg/mL of Human/Mouse/Rat Phospho-GSK-3a (S21) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4125), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho-GSK-3a (S21) at approximately 51 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

GSK‑3 alpha in Human Thymus.

GSK‑3a was detected in immersion fixed paraffin-embedded sections of human thymus using 5 µg/mL Human/Mouse/Rat Phospho-GSK‑3a (S21) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4125) overnight at 4 °C. Before incubation with the primary antibody tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained with the Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS005) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.