Anti muc1

Whole cell lysates were prepared as previously described (18 (link)). Samples were separated by SDS-polyacrylamide gel electrophoresis and electro-transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked with 5% dry skim milk and treated with anti-Phospho-Stat3 (Tyr705), anti-Stat3 (Cell Signaling Technology, Beverly, MA, USA), anti-Oct4 (BD Biosciences), anti-MUC1 (Abcam), anti-Bcl2, anti-Mcl1, anti-GAPDH, and anti-β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) antibodies. Immunoreactive proteins were visualized by horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc.) and an ECL reagent (ATTO Corp., Tokyo, Japan).

Immunofluorescence staining of Muc1 and Muc4 proteins was performed to validate the optimal fixation method determined above. Paraffin‐embedded sections (four serial sections per mouse, four mice per cutting orientation) were deparaffinized, rehydrated, and stained using a standard IHC protocol.²¹ Antigen retrieval was performed by heating sections in sodium citrate pH = 6 at 80°C water bath for 2 hours. Primary antibodies were anti‐Muc1 and anti‐Muc4 diluted at 1:100 (both Abcam, Cambridge). Secondary antibody was Cy 3 AffiniPure F(ab′)₂ Fragment Goat Anti‐Mouse IgG (H + L; Jackson ImmunoResearch Inc., West Grove, Pennsylvania) diluted at 1:200. Sections were incubated with the primary antibodies at 4°C overnight followed with secondary antibodies for 1.5 hours. Slides were mounted using Vectashield with DAPI (Vector Laboratories, Burlingame, California). Cryosections were air‐dried for 2 hours, washed with PBS (2 × 5 min), fixed with formalin, PFA, or acetone as mentioned above, then stained with Muc1 or Muc4 following the same protocol above. Images were taken with the Nikon Eclipse Ti2 inverted microscope with a DS‐Qi2 high‐sensitivity monochrome camera (Nikon Instruments Inc., Melville, New York) and adjusted for brightness using the NIS Elements software.