Hi Jess!

So, I haven't imaged through gelatin before but I have imaged cells through other similar hydrogels. Gelatin should be reasonably transparent as long as you use it at the correct concentration and so long as the gel is thin enough (less than 1cm). 

You can image without degrading the gel but I would recommend doing this using confocal microscopy. If you don't have access to a confocal you can still image but you can only really image the gel surface.

Another option is to treat your gels like tissue samples and get them sectioned; you'll get some really nice images that way too.

My main piece of advice if using antibodies with gels though, is that everything requires a lot more time. You'll have to leave the antibodies on the gel a lot longer than the protocols suggest, to ensure they penetrate the gel and you'll need to ensure you add extra washing steps, as antibodies can readily be trapped within the gels. Also, yes dead GFP cells will still fluoresce.

Hope that helps!

Laura

Try a confocal microscope, it should work! goodluck!

Hello! Interesting discussion, may I ask about my experiments?

I am about to test 2 things with immunofluorescence:

1) I have fibroblasts growing on 0.2% gelatin coated cover slips to perform IF. I have tried once and it seems there is debris or so all over the place and it is difficult to see the cells. I think is due to the coating (I use to coat with 0.2% gelatin for 1h at 37°C, wash, then dry in the hood for 2h. store for up to 2 weeks at RT. Wash with medium before use). I previously coated coverslips with 5µg/mL collagen and I did not have any problems (but I used the coated coverslips immediately without drying)... any suggestions?

2) I will soon stain organoids embedded in matrigel. I have been told that fixation with 4% PFA usually breaks down the matrigel dome and following a regular IF protocol is possible. Any experience with that? Should I increase the incubation times?

Many thanks!