I dissected tissue from mice then embedded it in matrigel for culture. For analysis, I plan to do immunostaining with some cell markers. But the regular IF protocol did not work.
Matrigel would be solid at room-temperature and dissolve in 4 ℃. Tissue was floating in buffer during antibody incubation and washing steps.
Since it is 3D culture, I could not mount them in the end and there were no signals....
Does anyone have suggestions?
Many thanks in advance.
Try doing IF at 4 degree. Keep for overnight incubation in 4 degree. Should immediately wash after you remove specimen out of plus 4. I guess this should work.
Could you perhaps provide more details on the protocol (fixation, incubation times, chemicals, temperature, etc) for troubleshooting?
How thick/big were your samples? What type of microscope are you using?
I did not quite understand if the free-floating staining was a problem for you or not. If you're using a confocal you do not necessarily need to mount the samples, there are for example 12-well plates with transparent bottom and black walls for imaging.
No signal sounds like the antibody binding did not work. Have you been using them on mouse tissue before? Perhaps longer incubation times or adding Triton-X for permeabilization might help.
Mix the 3D cells with OCT and freeze. Then cryosection it and follow IHC for cryosections. Good luck.