Long-Term Fixation

[SpaceMan]

I need to develop a protocol to fix hemocyte extractions from Drosophila. The samples need to last at least two weeks since immediate analysis is not possible. Also, I will be using LacZ, so Beta-Gal activity must also be decently preserved. I will also be doing GFP analysis--if fluorescence is disrupted, I can use an antibody, but then the GFP antigenicity must also be decently preserved. I know Glutaraldehyde blocks Beta-Gal, but is better for long term fixing than paraformaldehyde. Any glutar:paraform ratio suggestions? Do I need to permeablize the cells?

Thank you so much,
Matt

[adi]

hi,

let me begin by saying that i don't have an exact answer to any of your questions..only some suggestions that might work.

1. i've preserved GFP flouroscence in 4% PFA for approx 6 days. what are you fixing your cells with now and at what temp do you store them? 4 deg is a mighty good idea.

2. permeablization is a problem that has got a lot to do with the location of the GFP...cytosolic....you may have to permeabilize to get the antibody through in that case... ice cold methanol and 5 mins incubation at -20 will do admirably. membrane bound and exposed to the outside ..no need for permeablization.

FYI.....I've found that both glut and PFA cause a certain amount of permeabliation for all their much touted virtues.

a friend has tried gut:PFA = 1:1 ....but i can't vouch for how long it'll preserve samples.

hope this helps

best
adi