19.06.2024

After stumbling into Meng in lab V128, we discussed possibilities of stamping our micropatterns instead of stenciling them, as stenciling is clearly not working with the current protocol that I have been using. Therefore, I got inspired to try stamping, and this was the protocol I followed (Meng's protocol for stamping on polymer bottom dishes):

1. Do not treat PDMS stamps using plasma cleaner to make them hydrophilic. Simply add fibronectin on top, and let it sit for ca an hour (not sure if Meng lets it sit for an hour, but I did. Actually I left it adhere for 2 hours since I was doing fluorescent imaging with Silja). I was of course very curious as to what would happen if we would treat PDMS stamps with plasma cleaner and do make them hydrophilic, as this is what we have been doing with Silja for her protocol. So naturally, I made two sets of stamps - ones that were not treated, and ones that were.
2. I then added ca 100ul fibronectin on the stamps. The ones that were treated hydrophilically had the fibronectin spread across the surface of the stamp very well. The ones that were not had the fibronectin collected into a dot, so I had to make sure to add fibronectin to add all of the stamps. This was difficult, as the PDMS stamp was acting very hydrophobic towards the fibronectin.
3. I used two different types of fibronectin - one that was prepped by Silja (pure GFP fibronectin, higher concentration of GFP) and one that I had prepared by mixing in 1/10 of fibronectin volume of GFP fibronectin (so mixture of pure fibronectin, and some GFP fluorescent fibronectin). This should not make much difference except for how well we can see the patterns under the microscope. I should have used the same type of fibronectin, but I did not have both available to me at the moment, and since this is just for test purposes, I went ahead with the fibronectin that I already had thawed in the fridge.
4. After 2 hours, I blew away the rest of fibronectin from all the stamps, and washed the stamps with type 2 distilled water. I then blew the water off the stamps again, and they were ready to be stamped onto the Ibidi polymer-bottom petri dishes!
5. Before stamping, I treated the Ibiidi polymer-bottom petri dishes to be hydrophilic. This is something that Meng said I should do, so I did it. Originally the petri dishes are untreated, so they are hydrophobic.
6. Last step was to stamp the patterns and cover the petri dishes with aluminum foil and leave them in the fridge overnight to adhere. I do not think that it is necessary to leave them adhere for 24 hours, but I did not have time to check on them one hour later after stamping, so first thing that I will do tomorrow morning is look at the stamps under the fluorescent microscope.