immunostaining of 3D tissue followed by dissociation - (Jul/28/2015 )

Hello Everyone,

I need help. I'm performing the immunostaining of a tissue "in situ" (directly on the transwell). At the end of the process the immunostaining (nuclear marker) looks great. Next, I want to harvest the cells from the transwell and place the cells into a number of wells in a 96-well plate for an automated microscopy scoring. The problem is that the dissociation buffer didn't work very well and when I used tripsine the signal (secondary antibody) was lost.

Any ideas? I was going to try a scrapper but that can also damage the cells' membranes and I could also loose the signal. Any dissociation buffer products that you could recommend?

Thanks,

I guess trypsin is too harsh. Maybe accutase works, but this is just a suggestion. Accutase is not as strong as trypsin, and works with most FACS applications.