paraformaldehyde fixation - (Oct/30/2008 )
Hi,
My collegue gave me Immunostaining protocol for a protein but I'm not sure of the fixation step,
he used 2 concentrations of paraformaldehyde first was 2% for 10 min at 4C and the second concentration was 8% for 20 min at room temp. is this step accurate or not?? 
2% paraformaldehyde for 10 min at room
wash
8% parafromaldehyde
wash
Triton x-100 (0.1%)
wash
-Nadine.-
It depends a bit on what you are doing - most tissue lumps (approx 5 mm3) will fix well in 4% para within an hour, but if you are doing cells grown on a slide will fix well in 2% para within 15 minutes.
8% seems a bit strong to me, and if you are using it you probably don't need the 2% step.
-bob1-
QUOTE (bob1 @ Oct 30 2008, 04:20 PM)
It depends a bit on what you are doing - most tissue lumps (approx 5 mm3) will fix well in 4% para within an hour, but if you are doing cells grown on a slide will fix well in 2% para within 15 minutes.
8% seems a bit strong to me, and if you are using it you probably don't need the 2% step.
8% seems a bit strong to me, and if you are using it you probably don't need the 2% step.
I'm growing my cells(( from cell culture)) on coverslip.
-Nadine.-
QUOTE (Nadine. @ Nov 1 2008, 04:21 AM)
QUOTE (bob1 @ Oct 30 2008, 04:20 PM)
It depends a bit on what you are doing - most tissue lumps (approx 5 mm3) will fix well in 4% para within an hour, but if you are doing cells grown on a slide will fix well in 2% para within 15 minutes.
8% seems a bit strong to me, and if you are using it you probably don't need the 2% step.
8% seems a bit strong to me, and if you are using it you probably don't need the 2% step.
I'm growing my cells(( from cell culture)) on coverslip.
The 8% sounds redundant to me, but maybe special cell line or protein or such. Why don't you ask your colleague what is the purpose for that step?
-Almasy-
I fix my cells on coverslips with cold 3.7% formaledehyde (Fromalin) for 10-15 minutes at 4c.
-Curtis-