Dapi flour mount g

Formalin-fixed, paraffin-embedded tumor tissue sections were stained with various antibody for IF. Briefly, the paraffin was removed by Histo-Clear (National Diagnostics, GA, USA). For hydration, the slides were dipped in a gradient of ethanol solution (from 100 to 30%) and in water for 15 min at room temperature. Next, the slides were placed in 0.01 M citric acid buffer (pH 6.0) at 95 °C for 30 min for antigen retrieval. The slides were blocked for 1 h with 5% of goat serum in order to prevent the nonspecific binding of the antibodies and then treated with different specific primary antibodies: Ki67 (Abcam, Cambridge, UK), p-p65 (Cell Signaling, MA, USA), and F4/80 (eBioscience, CA, USA) for overnight at 4 °C followed by incubation with the secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG (H + L) and goat anti-rat IgG2a-FITC (Invitrogen, CA, USA) for 1 h at room temperature). Nuclei were counter-stained by DAPI Flour mount-G (SouthernBiotech, AL, USA). Images were then captured at least four sections of each samples using a fluorescence microscope (Leica, Germany) and at least four different samples per each group were analyzed by ImageJ program using ImageJ (fluorescent intensity area/total image area × 100) in each experiment.

The BMELs were treated 2 h prior to fixing with Brefeldin A (10ug/mL,Tocris 1231), an inhibitor of protein translocation to Golgi, to retain OPN inside the cells. The cells were fixed with 4% w/v PFA in 1XPBS solution at 72-h timepoint starting after the 6 h seeding time. This was followed by a 10-min permeabilization step using 0.25%TritionX/1X PBS solution and a 45-min blocking step with the blocking buffer (5% donkey serum in 0.25%TritonX/1X PBS solution). Both the primary and secondary antibody staining was either done overnight at 4 °C or for 1 h at room temperature in dark, with 3x 5 min 1X PBS washes in the middle. The antibody cocktail was prepared in the blocking buffer. Primary antibody anti-HNF4a (ab41898) was used at 1:200 dilution, anti-OPN (AF808) was used at 1:50 dilution and anti-E-Cad (AF748-SP) was used at 1:50. Actin was stained using fluorescent antibody Acti-Stain (PHDH1-A) at 1:700 dilution. All secondary antibodies (anti-mouse: ab98795, anti-goat: ab96935) were used at 1:50 dilution. For 12 mm coverslips, 30 µL of antibody cocktail was used to stain. Samples were then mounted in DAPI Flourmount G (Southern Biotech 0100-20).