Xt ultraview universal dab detection kit

The spheres and the clinical lung cancer specimens were embedded in paraffin blocks, and cut into 5-μm-thick sections. The sections were deparaffinized and stained with Hematoxylin and Eosin (HE), anti-αSMA mouse monoclonal antibody (Clone: 1A4, dilution 1:50, Dako), anti-human cytokeratin 7 (CK7) mouse monoclonal antibody (Clone: OV-TL 12/30, dilution 1:50, Dako), anti-CD31 mouse monoclonal antibody (Clone: JC70A, dilution 1:50, Dako) and anti-Ki-67 mouse monoclonal antibody (Clone: MIB-1, dilution 1:50, Dako). Immunohistochemistry was performed using a Benchmark XT (Roche) autostainer with the XT UltraView Universal DAB Detection Kit (Ventana Medical Systems, Inc.). We calculated the proportion of Ki67- and αSMA-positive cells by counting the immunohistochemically-positive cells and the total number of nuclei in three microscopic high power fields (HPF).

The entire transwell was fixed in 10% buffered formalin, and then the membrane was cut from the well using a scalpel and embedded in paraffin vertically. Immunohistochemistry was performed using the Benchmark XT autostainer (Roche) with an XT ultraView Universal DAB Detection Kit (Ventana Medical Systems, Tucson, AZ, USA), according to the manufacturers’ protocols. The images were obtained with a BZ-X700.
For preparation of cryosections, the fixed membrane was embedded in Tissue-tek O.C.T (Sakura Finetek USA, Torrance, CA, USA) and frozen at −30 °C.