Alexa fluor 594 goat anti rabbit immunoglobulin g igg

PDAC cells were treated with co-delivery NP, HCQ NP, or PAL NP for 72 h at equivalent drug concentration (PAL = 2.5 μM, HCQ = 10.8 μM). PANC-1 cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 3 wt% bovine serum albumin (BSA). Primary antibodies included anti-phospho-Rb (1:1000, cat#8516, Cell Signaling Technology), anti-p62 (1:100, cat#ab91526, Abcam), anti-Ki67 (1:500, cat#ab15580, Abcam), anti-CC-3 (1:400, cat#9664, Cell Signaling Technology), anti-Bcl-2 (1:150, cat#ab182858, Abcam), anti-Mcl-1 (1:500, cat#ab32087, Abcam), or anti-Bcl-x_L (1:500, cat#ab32370, Abcam) accordingly. Cells were further incubated with secondary antibodies, including Alexa Fluor 594 goat anti-rabbit immunoglobulin G (IgG; 1:500, cat#A11012, Thermo Fisher Scientific) or Alexa Fluor 488 goat anti-rabbit IgG (1:500, cat#A11008, Thermo Fisher Scientific), and counterstained with 4,6-diamidino-2-phenylindole. The immunofluorescence staining was visualized by confocal microscopy. Fluorescence intensity of biomarkers including Ki67, Bcl-x_L, Mcl-1, and Bcl-2 was quantified with the ImageJ software (v1.5.2) and normalized to the number of cells as indicated by nuclei stain.

Cultured cells grown to 50% confluency on coverslips were serum-deprived for 24 h and then treated for 2 h with FGF2 alone and in combination with the FGFR1 inhibitor PD173074, the MEK inhibitor PD98059, or PI3K inhibitor Wortmannin, as indicated. Thereafter, cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated overnight with a rabbit primary antibody against c-Rel (#4727) (Cell Signaling, Euroclone, Milan, Italy). After incubation, the slides were extensively washed with PBS, probed with Alexa Fluor 594 goat anti-rabbit immunoglobulin G (IgG) (1:300, Thermo Fisher Scientific, Monza, Italy) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:1000; Sigma-Aldrich, Milan, Italy). Then, the slides were imaged on the Cytation 3 Cell Imaging Multimode reader (BioTek, AHSI, Milan Italy) and analyzed using the software Gen5 (BioTek, AHSI, Milan Italy).