Dako target retrieval solution s1700

IF was performed as previously described [69 (link)] after tissue deparaffinization by clearance in xylene and hydration through graded ethanol series. Antigen retrieval was conducted at 99°C in Dako Target retrieval solution (S1700) for 20 min per manufacturer’s instructions (Agilent Technologies, Copenhagen, Denmark). Washes were performed in IF buffer (130 mM NaCl, 7 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, 7.7 mM NaN₃, 0.1% bovine albumin, 0.2% Triton X-100, 0.5% Tween-20). Due to use of mouse antibodies on mouse tissue, blocking and antibody dilution were performed using the Mouse on Mouse (MOM^™) Kit following manufacturer’s instructions (Vector Labs, Burlingame, CA). Sections were stained using human antibodies targeted against Keratin 19 (Neomarkers #MS-198-P1), anti- actin smooth muscle (Spring Biosciences #E2464), anti-ki67 (Dako #M7240), or anti-IFITM1 (Santa Cruz Technology). Secondary antibodies were FITC or Texas Red conjugated (Santa Cruz). Sections were mounted using ProLong^® Gold Antifade Reagent with DAPI (Cell Signaling) and visualized on a Leica TCS SPE confocal microscope in the Confocal Imaging Core at The University of Kansas Medical Center. Images were collected and analyzed using the Leica LAS AF Lite software (Leica Biosystems, Nussloch, Germany). For quantification, mean fluorescent intensity was determined using Image J software on single color channel images.

IHC staining was performed after tissue deparaffinization by clearance in xylene and hydration through graded ethanol series. Antigen retrieval was conducted at 99°C in Dako Target retrieval solution (S1700) for 20 min per manufacturer’s instructions (Agilent Technologies, Copenhagen, Denmark). For human samples, blocking was performed using 5% normal horse serum and antibody dilution was performed in 0.01% Triton-X. For mouse xenografts, blocking and antibody dilution were performed using the Mouse on Mouse (MOM^™) Kit following manufacturer’s instructions (Vector Labs, Burlingame, CA). Sections were stained using primary human antibodies targeted against IFITM1, ERα, phospo-STAT1 (ser701), CD31, MMP1 (Santa Cruz) and Ki67 (Dako) and HRP-conjugated biotinylated secondary antibodies (Vector Labs). Immunoperoxidase signal was produced using 3,3′-Diaminobenzidine (DAB) and amplified using the Vectastain^® Elite ABC Kit (Vector Laboratories). Tissue sections were counter stained using hematoxylin and mounted in xylene. Slides were imaged on a Nikon Eclipse 80i Upright Microscope in the Imaging Core of KUMC.