[Histonet] ER labelling of fixed cells

[James S.]

Hi all,

I'm trying to do some double labelling to show that a protein resides in the ER but I'm having some trouble getting nice reticulated ER staining in fixed cells. So far I have tried fixing in 4% PFA (15min on ice) followed by permeabilization with Tritonx100 (0.25%; 10min; room temp) or Saponin (0.5%; 7min; room temp; all susequent solutions containing 0.05% saponin). ER antibody is rabbit anti Calnexin (Stressgen).
The labelling I'm getting is quite strong but is very punctate. Staining of my protein of interest is also very puntate but doesn't appear to colocalise with the clanexin and also I don't see any staining of the plasm membrane - which I should! Anyway the Prof wants to see nice reticulated staining of the ER - which I have seen in live cells with ER tracker but is it possible to get the same thing with fixed cells?
I thought about trying to fix with glutaraldehyde but ?concentration ?time......
Also my PFA was in PBS would it be better to put it in HEPES or PIPES?

Any suggestions welcome!

Thanks
Sonya