Kaiser s glycerin gelatin

To assess the effect of DUSP1 silencing on translocalization of STAT1, cells expressing LV-cont or LV-shDUSP1 were seeded at a density of 2 × 10⁵ cells per plate. After 1 day of seeding, cells were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS for 5 min, and incubated at 4°C overnight with a 1:50 dilution of anti-STAT1 (Cell Signaling Technology, Beverly, MA) in blocking solution (1% bovine serum albumin). Cells were washed three times with PBS, then incubated with Alexa 594-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 h at room temperature. Nuclei were detected by staining with 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 min. After three washes with PBS, preparations were mounted with Kaiser’s glycerin gelatin (Merck, Darmstadt, Germany) and examined using an Axiovert fluorescence microscope (Carl Zeiss, Göttingen, Germany).

Briefly, transwell inserts containing membranes with 8 μm pore size (Nunc) were coated with matrigel (Cat N° 356231, growth factor reduced matrigel matrix, phenol red free, BD Biosciences) as per the manufacturer's instructions. Cells (75,000) were spread over the matrigel (1:5) in 200 μl of medium with no serum, and 800 μl of culture medium with 5% of serum was added to the lower chamber. Cell invasion was studied under the effect of platelets, metformin or both. These treatments were added on the lower chamber of the invasion well. For assessing the number of invaded cells, the filters were fixed with PFA for 30 minutes at room temperature and stained with crystal violet. The filters were mounted on cover slides with Kaiser’s, glycerin-gelatin (Merck). Each experiment was performed in duplicate and was repeated at least 3 times. The inserts were examined using a microscope (Nikon Eclipse E200) with attached camera; photographs of 10 fields at 40X, were obtained per insert, and the number of cells in the underside of the filter was counted. Results were expressed as cell number per field, corresponding to the average of the cells photographed for the correspondent treatment.

Tissue for staining was stored at −80 °C and cut into 5 μm cryosections. The sections were air-dried for 30 min and fixated for 10 min in acetone. Washing and blocking of aspecific binding sites, endogenous peroxidases, and endogenous biotin was performed according to standard procedures. The sections were incubated for 60 min at RT with primary antibody (see Additional file 2: Table S2), and with species specific HRP- or biotinylated secondary antibodies (DAKO and Southern Biotech). Stainings were visualized using VECTOR Red Alkaline Phosphatase (AP) Substrate or ImmPACT DAB Peroxidase (SK5100 or SK4105, respectively, Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. All immunohistochemical stainings were counterstained with hematoxylin (Merck, Darmstadt, Germany) and mounted in Kaiser’s glycerin-gelatin (Merck).