It depends upon the markers you are wishing to detect. Some are wrecked pretty quickly - so it's best to fix for even only a few minutes. Other markers are more robust and will tolerate being fixed for days. If you're worried about over fixing, fix at 4 degrees, in the fridge, with a lower concentration of formalin. If they are fixed tissues, I transfer to 70% ethanol where they can remain for days or weeks.

Dear Aleksandra, Simon gave you a very good answer. The best policy for fixation, especially with aldehydes is to choose the shortest possible time - in case of cells many publications quote 10 minutes. For tissues - time of fixation depends on the size and type of tissue (if e.g. the method taken from the literature calls for 4 - 6 hours at room temperature, then it can be slowed down and kept in the fridge at 4deg C in the fridge

Hi Aeksandra,

Both Maria and Simon have gave excellent suggestion. If you are making cell smears on slides, fix with 4% paraformaldehyde for 10-15 mins, then give 3 washes with PBS and store your slide in a moist chamber at 4 deg. For tissues you need to extent your fixation time to several hours but at a lower temperature.

Regards,

Prasad.

Dear Aleksandra,

For my cell cultures, I fix them for 5-10 min. in ice-cold acetone and then store them in the -80°C in aluminium foil. You can store them there for several years if needed. It gives very nice IF staining. Lately, i used cell cultures fixed in acetone and stored for 12 months in the -80°C and the stainings were very pretty using golgi staining, ER staining etc. In a general manner, we keep our cell cultures and tissues fixed or non-fixed in the -80°C. To avoid freezing artefacts, keep your slides after thawing very hydrated in moist chambers during staining protocol. Make sure to spin off twice your secundary IF antibody to avoid debris in your pictures. It's another way than the 4°C method, but i'm very happy with the results.

Good luck!

Sandrine

You also want to avoid storing in formalin, because over time formalin oxidizes to form formic acid, which will substantially degrade your histology and immunostaining.  This is the same reason why you want to use fresh formalin/PFA, as once the fixative is exposed to air it will begin degrading.

As others pointed out, 70% EtOH is a relatively good storing solution for a couple weeks.  70% EtOH is sort of in a sweet spot, where neither polar material like soluble proteins will dissolve into solution, and neither will non-polar lipids and membrane bound proteins.  But, in reality, both still slowly go into solution ,just in relatively equal proportions, meaning the longer the storage in EtOH, the worse the histology will be.

If you are trying to store cells because you are doing an experiment at different time points,  the better approach is to sequentially start your experiments, so that the end point is all on the same day, so you can do all of your staining at the same time.

This is important, because a sample that has been stored in EtOH for a week cannot be compared to a fresh sample, as they are apples and oranges (i.e. one was stained immediately, the other was allowed to degrade for some time).  If you need to compare samples, they need to have identical fixation and staining procedures performed, ideally simultaneously.