Millicell ez slides eight well glass slides

To assess the ability of resveratrol to induce the apoptotic process, an ICC reaction was performed. The cells of the tested lines were plated on Millicell EZ SLIDES eight-well glass slides (Merck Millipore, Gernsheim, Germany) in the following amounts: EPP85-181P—1 × 10⁴ cells/well, EPP85-181RNOV—1 × 10⁴ cells/well, AsPC-1—1.5 × 10⁴ cells/well and H6c7—1.5 × 10⁴ cells/well. 24 h later, cells were treated with resveratrol at concentrations of 0, 25, 50 and 100 µM for 48 h. After this time, cells were fixed with methanol-acetone (1:1) for 10 min at 4 °C. The ICC reaction was performed on an Autostainer Link48 (Dako, Glostrup, Denmark). The following primary antibodies were used: Bcl-2 (Dako, Glostrup, Denmark), Bax (Santa Cruz Biotechnology, Dallas, TX, USA) and activated Caspase-3 (Cell Signaling Technology, Boston, MA, USA). Slides were first incubated with primary antibodies against Bcl-2 (ready-to-use), Bax (1:25) and activated Caspase-3 (1:400) for 20 min at room temperature, followed by 20 min with EnVision FLEX/HRP (Dako, Glostrup, Denmark). In the next step, the slides were incubated for 10 min with 3,3’-diaminobenzidine (DAB, Dako. Glostrup, Denmark). The slides were counterstained with EnVision FLEX Hematoxylin (Dako, Glostrup, Denmark) and sealed with coverslips in a mounting medium. The ICC reaction was assessed using a BX-41 light microscope (Olympus, Tokyo, Japan).

For immunofluorescence, AsPC-1 cells were plated (1.5 × 10⁴ cells/well) on Millicell^® EZ SLIDES eight-well glass slides (Merck Millipore, Gernsheim, Germany). After 24 h, cells were treated with resveratrol (0 and 100 µM) for 48 h. Cells were then fixed in 4% paraformaldehyde (12 min, room temperature). The membranes were permeabilized with 0.2% Triton X-100 (10 min, room temperature). Nonspecific binding sites were blocked with 3% BSA in PBS (1 h, room temperature). Cells were incubated overnight at 4 °C with primary antibodies: Bax (1:25 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) in 3% BSA/PBS and Bcl-2 (ready-to-use, Dako, Glostrup, Denmark). Protein detection was performed with Alexa Fluor 488 conjugated antibody secondary antimouse (dilution 1:2000, Abcam, Cambridge, UK, Cat# ab150113, RRID: AB_2756499), incubation 1 h, temp. 4 °C. The slides were sealed in a medium containing DAPI (Invitrogen, Carlsbad, CA, USA). The analysis of the proteins levels was performed using a Fluoview FV3000 confocal microscope (Olympus, Tokyo, Japan, RRID: SCR_017015) with the cellSens software (Olympus, Tokyo, Japan, RRID: SCR_016238).