Eclipsf 90i microscope

Panc-1 cells, seeded in chamber slide dishes, were pre-incubated for 60 min in the presence of PA 1×IC₅₀, erlotinib 40 nM or equivalent volumes of DMSO. Following stimulation with EGF 10 ng/ml for 15 minutes, Panc-1 cells were fixed with paraformaldehyde (4% w/v in PBS). In a second experiment, Panc-1 cells, MIA PaCa-2 cells and AsPC-1 cells were treated with PA 1×IC₅₀ for 4h. Immunostaining was performed for p-EGFR (Cat # ab40815; Abcam, Cambridge, UK). After PBS washing, sections were incubated with an Alexa Fluor 488-linked secondary antibody. After staining, slides were mounted in VECTASHIELD with DAPI and photographed under a Nikon ECLIPSF 90i microscope with a digital camera.

Panc-1 cells, seeded overnight in chamber slide dishes, were treated with CMC2.24 1x and 1.5x-IC₅₀ for 4 h. Following the treatment, Panc-1 cells were washed three times with PBS and fixed with paraformaldehyde (4% w/v in PBS). Immunostaining was performed using rabbit anti-p-ERK or p-STAT3 antibodies. After PBS washing, sections were incubated with a fluorophore-linked secondary antibody (Alexa Fluor 488–anti-rabbit IgG; Life Technologies). After staining, slides were mounted in VECTASHIELD with DAPI (VECTOR Laboratories, Burlingame, CA) and photographed under a Nikon ECLIPSF 90i microscope with a digital camera.