Hello Karin,

this mainly depends on how big your GFP-containing protein is. The main problem with the 'standard' fixation of free GFP is that it leaks out of the cells before the fixation is completed.

We use Heinen et al. Cytometry 2014 (PMID: 24616430) as reference.

Good luck,

Gerhard

These are CHO not mice cells, but Caltag Fix&Perm seemed to work fine for GFP here.

"For intracellular staining, cells were trypsinized,

washed in cold PBS and re-suspended in PBS at a concentration

of 1
106 per 100 ml. An equal volume of Caltag

Fix&Perm (Invitrogen) solution A was added and cells

were incubated for 15 min. Cells were washed once with

PBS containing 5% normal goat serum and re-suspended

in 100 ml of Caltag Fix&Perm solution B. For each sample,

250 ng of rabbit polyclonal anti-GFP (ab6556; Abcam,

Cambridge, MA, USA) antibody or rabbit polyclonal antihuman

C reactive protein antibody (A-0073; Dako, Ely,

UK) labelled with the Zenon rabbit immunoglobulin

G Alexa647 kit fromInvitrogen was added. Cells were incubated

in the dark for 20 min, washed with PBS containing

5% normal goat serum (Abcam) and analyzed immediately."

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