I am planning a time-course experiment where my organotypic slices will be stained after different periods in culture. In a ideal world, I would like to do this all at once but since my cultures are staggered over several days, I was wondering if anyone knew how long tissue (brain slices) can be kept/stored prior to antibody incubation so that I can do the whole technique all at once.
My protocol is to fix in PFA for 10 min at RT, 20% MeOH wash, and permeabilise in 0.5% TX-100 in PBS for 12 hours minimum before proceeding to dual stain.
Thanks in advance.
Dear David,
there are very few antigens that are sensitive to prolonged storage. Most, however, are not. For a straightforward experiment I would recommend to fix as you do, then leave in buffer at 4C (do not extend fixation times, rather change washing buffers several times as proposed by Kristin). Start all subsequent steps (permeabilisation etc.) only when you have collected all tissues.
Provided you have worked out your double staining protocol beforehand; I would be surprised if this does not work well.
Best of success, Amin
Thanks guys.
Kristin: I'm using ice-cold 4% PFA in PBS.
Roxana, I had a feeling that using an inert solution with azide would be the best option. Do you have a protocol?
So if I fix, wash 3x 5 min in PBS then store the slices in PBS + 0.01/0.02% sodium azide, do you reckon they'll be ok for 10 days at the most if kept in the fridge? (my last timepoint is 10 DIV). Then proceed with MeOH wash and so on...
If Your material is constituted by slices of brain (200-500 micrometers?), a PF 4% needs a short fixation time (1/2 hr to 2 hrs) with a good preservation of antigenic sites for histochemistry. As a general rule, a short fixation time gives a higher probability of good preservation of antigenic sites for histochemistry, even if the morphology will be not so good. A long fixation time will give a good morphology, but the probability of modifications of the molecular sites for histochemistry will be higher. Synthesis: Short fixation time=Good histochemistry. Best wishes!
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