Last Updated on April 8, 2022
Fluorescent staining after mechanotransduction.
Important qualitative and quantitative observations can be made about cell signaling with the effective application of cell fluorescent staining techniques. Along with cytoskeletal and morphological changes.
A driving goal behind almost every investigation in mechanotransduction is the cells’ biochemical response to mechanical stimulation.
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 use to ensure conclusive results. Here is a popular option we have found.
Step by step instructions for fluorescent staining
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 tool. A 0.8 x 0.5 cm sized piece usually works well, but greater surface area excisions are possible with our larger chambers (STB-CH-10). We recommend cutting the membrane in a rectangular pattern to recall which direction the stretch was applied. This is to avoid 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 it with a mounting medium. We have seen successful tests with the PermaFluor™ product in the past. Your slide is now ready for transfer to a fluorescent microscope for observation.
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