A driving goal behind nearly every investigation in mechanotransduction is the cells’ biochemical response to mechanical stimulation. Along with cytoskeletal and morphological change, important qualitative and quantitative observations can be made about cell signaling with the effective application of cell staining techniques. The flexible membrane of the PDMS can present new challenges though, especially compared to the commonly used static glass slide these stains are traditionally performed on. There are a couple of techniques one can employ to ensure conclusive results. Here is a popular option we have found.
Once the stretching of cells on the PDMS membrane has concluded, cut out a piece of the PDMS, with ECM coating and cells still on it, using a surgical knife or similar implement. A 0.8 x 0.5cm sized piece usually works well, but greater surface area excisions are possible with our larger chambers (STB-CH-10). It is recommended to cut the membrane in a rectangular pattern to be able to recall which direction stretch was applied without compromising the quality of the sample with a label.
The piece of membrane should then be placed within a container of suitable size in PBS. Wash the membrane and cellular material with PBS twice. After washing, fix with 4% formalin solution in PBS at room temperature for 5 minutes and gently shake. Then wash again with PBS 3 times over 5 minutes. Introduce 0.1% TritonX-100 in PBS to pierce the cell membrane and shake for another 5 minutes. Execute another 3 times-wash with PBS for 5 minutes. Using a primary antibody in 0.1%Tween20 in PBS at room temperature, shake for 30 minutes. Wash 3 times again for 5 minutes with 0.1%Tween20 in PBS. Repeat the last 2 steps with the secondary antibody in 0.1%Tween20 in PBS at room temperature and shake for 30 minutes.
With the membrane prepared, place it cell-side down on a slide glass and enclose with a mounting medium. We have witnessed successful tests with the Perma Fluor product in the past. Your slide is now ready for transfer to a fluorescent microscope for observation.
Further protocols and techniques can be found on our website at StrexCell.com and product support and inquiries can be requested at any time at firstname.lastname@example.org.