Lsm image analysis software

Fine sections (4 μm) were prepared from formalin fixed paraffin embedded tumor tissues and fixed on glass slides (Safeline Histopathology, Pune, India). For immunohistochemistry, slides were deparaffinized by xylene solution twice for 10 min and subsequently rehydrated in graded alcohol (100%, 95%, 70% and 50%). For antigen retrieval, slides were boiled in sodium citrate buffer (0.01 M, pH 5) at 100 °C for 10 min and allowed to cool at room temperature. BSA (0.2%) was used for blocking for 1 hr. After washing with TBST, slides were probed with DKK4 (1:100) (Santa Cruz Biotechnology, CA, USA) and IHC specific β–catenin (1:100) antibody (BD Bioscience, NA, USA) and incubated at 4 °C overnight. Slides were washed with TBST and probed with compatible FITC or TRITC conjugated secondary antibody for 3 hr. After five washes with TBST, tissue sections were layered with mounting medium containing DAPI (Santa Cruz Biotechnology, CA, USA). Tissues were examined and images were captured with a confocal microscope at 60x magnification (LSM510 or META, Carl Zeiss). Images were subsequently processed by LSM image analysis software (Carl Zeiss, IL, USA).

HepG2 cells were seeded on multi-well chambered slides (MP Biomedicals, OH, USA). Cells were treated as mentioned in pertinent figure legends and processed for staining as described previously [27 (link)]. Cells were analyzed under Zeiss LSM 510 microscope, and images were processed by LSM image analysis software (Carl Zeiss, Germany).