Alexa fluor anti rabbit

Precision cut lung slices were incubated with primary antibodies: rat anti-mouse Ly-6G (clone 1A8, Bio X-Cell), hamster anti-mouse CD31 (2H8, Life Technologies), rat anti-mouse S100A9 (MU14-2A5, Hycult Biotech), polyclonal rabbit anti-mouse Ci-H3 (ABCAM) diluted in PBS-BSA-Triton-for 48h at 4°C. The sections were washed in PBS and incubated with low species cross-reactive fluorophore-conjugated secondary antibodies (Cy3 / Cy5 anti-rat, anti-hamster, Jackson; AlexaFluor anti-rabbit Life Technologies) for two hours. Sections were washed in PBS-BSA-Triton, then PBS and post-fixed with 4% PFA for 5 min. After a subsequent rinsing step, the slices were transferred into a 24-well imaging plate (IBIDI), covered with buffered Mowiol 4–88, pH 8.5 (Sigma-Aldrich) then coverslipped. Sections were evaluated by using a confocal laser-scanning microscope (TCS-SP5, Leica).

Fixed and permeabilized cells or tumor sections were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature, followed by incubation with ki67 antibody overnight at 4°C. After washing with PBST for 30 min, Alexa Fluor anti-rabbit (Life Technologies, OR, USA) antibody was applied for 1 h in the dark. Slides were mounted with ProLong Gold antifade reagent and DAPI. Images were obtained using a Zeiss fluorescence microscope.

Cells were crawling on slides followed by fixation and permeabilization. Samples were blocked with 3% BSA for 1 hour at room temperature, followed by incubation with E-cadherin (Cell Signaling Technology, Danvers, MA, USA) antibody overnight at 4 °C. After washing with PBST for 30 minutes, Alexa Fluor anti-rabbit (Life Technologies) antibody was applied followed by incubation for 1 hour protected from light. The slides were mounted with Prolong Gold antifade reagent with DAPI (Life Technologies). Pictures were obtained using a Zeiss fluorescence microscope.