Echo revolve hybrid microscope

Transwell assay was performed in 24-well inserts (8.0 μm; Corning). Briefly, 5 × 10 5 cells suspended in 200 μl serum-free medium were loaded on the top, and 500 μl complete medium was placed into the bottom side. 48 h later, the migrated cells were stained with 0.1% crystal violet. Images were captured at room temperature using Echo Revolve Hybrid Microscope (Echo) using a 10× objective at brightfield mode. Cells were counted using ImageJ software and the data were presented as the mean ± SEM., calculated from four randomly selected fields.

Paraffin-embedded tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Subsequently, the endogenous peroxidase activity of sections was blocked with 3% H 2 O 2 in methanol. After washing with 0.01 M PBS (15 mM NaCl, 8 mM Na 2 HPO 4 , and 2 mM NaH 2 PO 4 ) three times, the sections were blocked by 0.01 M PBS supplemented with 5% normal goat serum and 0.3% Triton X-100. Next, sections were incubated with primary antibodies against XAF1 (1:100; Affinity), RNF114 (1:100; Absci), and JUP (1:50; Cell Signaling Technology), respectively, overnight at 4°C. After washing with 0.01 M PBS three times, the sections were incubated with a secondary antibody (1:500) for 2 h. After washing once with 0.01 M PBS and twice with 0.05 M Tris-HCl (pH = 7.6) solution, the sections were visualized with 0.03% 3,3′-diaminobenzidine in 0.05 M Tris-HCl (pH 7.6) and counterstained with hematoxylin. Images were captured at room temperature using Echo Revolve Hybrid Microscope (Echo) using 20 × objective at bright field mode. The extent and staining intensity of protein were scored automatically by Vectra 2 system (PerkinElmer).