Fixation protocol depends upon the purpose of sections to be kept. Fixation in cold acetone (-20 c) for twenty minutes and then storage at -80 C will be good for immunoflorescence studies.

However fixation with methanol for 20 min/ ethanol for 20 min are also good.

Fixing in acetone is definitely the way to go.

I usually cut the sections, allow to dry and then put in the -80 for up to 6 months. But never allow these sections to thaw when taking them out of the freezer to retrieve sections from the same slide box. My lab always has a large cooler with dry ice or liquid nitrogen in it, which we put the slide box in to remove the needed sections. If the slides go through numerous freeze-thaw cycles the antigen and section will be damaged.

The fixative used depends on your antibody. I have had some antibodies work very well in acetone, whilst others give better staining in PFA. Of course, you also have to test the specificity of the antibody in the different fixatives as you can get false positives. Check the literature to make sure the staining is consistent with what is known, or if there is no information use transfected cells stained under the same conditions. I have had non-transfected cells stain with acetone fixation but not PFA which stained specifically the transfected cells.

Thank you,

How long do you normally leave the sections to dry before either fixing or storing in -80? I'm planning to mount on gelatin coated slides, but also have access to poly lysine and superfrost slides

I'm performing immunofluorescence and my plan was to optimise antibodies and also try the 3 different fixatives I mentioned above to get the best staining from each

Claire

I usually let them dry for at least 15 minutes but up to 1 hour is also fine. 

We usually use Superfrost slides to reduce issues of tissue detachment. I know they are more expensive but that wasn't a major issue for my lab in a pharma company. Previously in academia I used poly lysine coated slides to reduce costs and they were fine. I don't like gelatin coated as I found I had issues with background staining.

Just to clarify (I'm an IF newbie): If sections are cut and then stored, do they need to be fixed before they are stored in the -80? I plan on cutting frozen muscle biopsies and staining them within the next couple of weeks. Should I fix them and then put them in the -80? Would -20 be ok? My protocol calls for 4% PFA and this is for IF. Thanks!