Macrophage fixation for immunofluorescence - Sucrose yes or sucrose not (Aug/19/2008 )

Hi everybody again,

I've been trying to detect a membrane protein in murine peritoneal macrophages. The protocol for macrophage fixation is the next:

CELL PREPARATION
Coverslip with fibronectin 10ugr/ml 1h to overnigth at 37 degrees
Leave them o/n for attaching in RPMI medium (penic/Strept, 10mM Hepes, L-glut, and 10% FSB) 37 degrees
Wash not attached cell with HBSS at 37 degrees: twice
Fix attached cell with PFA 4% + Sucrose 2% during 5-6min at 37 degrees
Wash cells with warm-HBSS twice and stored them at 4 degrees

But when I stainned the cells I found a particular spot near the macrophage's nuclei (the circle in attached picture, the correct signal should be the square). I'm sure that spot is a artifact, but, could it be by fixation process (most certanly the sucrose use)?

Could some recommend me other fixation methods to preserve membrane GPI proteins in immunofluorescence techniques.

Thank you very much for your help

Anteros

I'm sorry but the image doesn't have the circle nor square, the artifact is the highest signal near the nucleus

Thank you very much for your help

Anteros
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ok, I just finished my first successful IF because I wasn't able to get sharp images before, and I can tell you that I have done 3 different fixation methods so far

1- Methanol for 10 min
2- Methanol 10 min, Aceton 10 min (aceton for permeabilization)
3- Formaldehyde 3.7% 15-20 min

some people also go for Paraformaldehyde. it's up to you.

for some reason Methanol+aceton gives me better images.