ICC of cell on coverslips - losing cells at permeabilization - (Dec/03/2006 )
I'm doing immunocytochemistry of neuroblastoma cells (such as SH-SY-5Y) on 13mm coverslips. I seem to be losing a lot of cells at the permeabilization stage. I fix in 4% paraformaldehyde for 15min on ice rinse in PBS and then permeabilize in 0.1% Triton for 10min on ice. When I rinse after the Triton, alot of cells are coming off the coverslip, despite best efforts to do this gently: dribbling the PBS down the side of the well, not squirting it on to the cells! Any suggestions for anything I can try?
-TeelaBrown-
QUOTE (TeelaBrown @ Dec 3 2006, 03:50 PM)
I'm doing immunocytochemistry of neuroblastoma cells (such as SH-SY-5Y) on 13mm coverslips. I seem to be losing a lot of cells at the permeabilization stage. I fix in 4% paraformaldehyde for 15min on ice rinse in PBS and then permeabilize in 0.1% Triton for 10min on ice. When I rinse after the Triton, alot of cells are coming off the coverslip, despite best efforts to do this gently: dribbling the PBS down the side of the well, not squirting it on to the cells! Any suggestions for anything I can try?
Try fixation at RT and use PBS supplemented with MgCl2 instead of PBS because it avoids cells detachment
-dnafactory-
QUOTE (dnafactory @ Dec 3 2006, 04:05 PM)
QUOTE (TeelaBrown @ Dec 3 2006, 03:50 PM)
I'm doing immunocytochemistry of neuroblastoma cells (such as SH-SY-5Y) on 13mm coverslips. I seem to be losing a lot of cells at the permeabilization stage. I fix in 4% paraformaldehyde for 15min on ice rinse in PBS and then permeabilize in 0.1% Triton for 10min on ice. When I rinse after the Triton, alot of cells are coming off the coverslip, despite best efforts to do this gently: dribbling the PBS down the side of the well, not squirting it on to the cells! Any suggestions for anything I can try?
Try fixation at RT and use PBS supplemented with MgCl2 instead of PBS because it avoids cells detachment
won´t it precipitate as Mg-phosphate? but I believe that bivalent cations may stabilize attachment
-The Bearer-