Prolong glass antifade mountant p36980

Immunofluorescence staining was achieved on PANC-1 and MIA PaCa-2 alone or stimulated with LIF 10 ng/mL, EC359 25 nM, and mifepristone 10 μM. Cells were plated on slides using cytospin. The spots obtained were fixed in methanol for 20 min and then were washed 3 times with phosphate buffered saline (PBS 1X).
Subsequently, cells were permeabilized and then incubated with blocking buffer (PBS 1X with 10% horse serum and 1% BSA) for 1 h at room temperature. Primary antibodies anti-LIF-R (ab235908), anti-Vimentin (ab92547), anti-Ki67 (ab16667) (Abcam, Cambridge, UK) were incubated overnight at 4 °C. The next day, after 3 washes with PBS 1X containing 0.1% Tween 20 (PBST), cells spotted were incubated with secondary antibody Goat Anti-Rabbit IgG H&L (Aleza Fluor 488) (ab150077) for LIF-R and vimentin and with Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (Alexa Fluor™ 568) (Invitrogen, Thermofisher scientific, Waltham, MA, USA) for Ki-67, for 1 h at room temperature in the dark. After 3 washes with PBST, the nucleus was counterstained with DAPI 1X for 1 min in the dark, and the reaction was stopped with a final wash in PBS 1X for 5 min. Then, slides were mounted with ProLong Glass Antifade Mountant (P36980) (Invitrogen, Thermofisher scientific, Waltham, MA, USA), sealed with nail polish, and observed with a fluorescence microscope Olympus BX60.

Immunofluorescence (IF) staining was carried out using MIA PaCa-2 cells. Cells cytospins were fixed in methanol for 20 min and then washed 3 times with phosphate buffered saline (PBS 1X), permeabilized and then incubated with Blocking buffer (PBS 1X with 10% horse serum and 1% BSA) for 1h at room temperature. Primary antibodies, anti- GPBAR1 (NBP2-23669), (Novus Biologicals) and anti-FXR (ORB156973) (Biorbyt) were dissolved in Blocking Buffer and incubated overnight at 4°. On the following day cells were washed three times with PBS 1X containing 0,1% Tween 20 (PBST), and then incubated with the secondary antibody, Goat anti-rabbit IgG (H + L) Alexa Fluor 488 (ab150077) (Abcam) for GPBAR1 and Goat anti-rabbit IgG (H + L) Alexa Fluor 568 (A11011) for FXR (Invitrogen), diluted in Blocking Buffer for 1h at room temperature in the dark. After 3 washes with PBST, nucleus was counterstained with DAPI 1X for 1 min in the dark and the reaction was stopped by a final wash in PBS 1X for 5 min. Then, slides were mounted with ProLong Glass Antifade Mountant (P36980) (Invitrogen, Thermofisher scientific Waltham, Massachusetts, USA), sealed with nail polish and observed at fluorescence microscope (Olympus BX60, Rome, Italy).