Hello
I'm doing a lot of immunofluorescence staining on kidney frozen sections. Currently, I have a lot of sections with a really bad structure whereas previously, sections of the same organ looks good (For the freezing, I put the kidneys in OCT, freeze in isopentane and store at -80°C until cutting).
What I'm doing actually is to keep the tissue at -80°C, put it at -20°C for cutting (10-15 sections) and go back at -80°C until the next session of cutting. So my tissue is doing a lot of going and coming at the -80°C freezer.
Someone knows if these numerous change of temperature can affect my tissue/organs?
Is it better to cut all my tissue in one time an to store the sections in freezer to avoid these thermal changes? And how long i can store acetone fixed sections in the freezer ?
Dear Romain,
I work with cryo-sections from brain but not from kidney. However, it shouldn't make a big difference. To my mind, repeated changes of temperature is very harmful for the tissues. I always cut the whole brain and store the slices at -20°C (-80°C is even better, however, we don't have enough space in the -80°C) until I fix and stain them. If you use the slices within a few weeks, there is nothing more to consider except that you shouldn't take out slices from the freezer if you don't process them afterwards. If you want to store them for a longer period of time, you should add silica gel pads to the box to avoid hoarfrost. I've never stored post-fixed slices, so I cannot give you any advices regarding that question.
Good luck!
Thanks for your answers !
Anne-Sophie, as you advise me, I will cut the entire kidney and store the sections until I use them. I just have to avoid hoarfrost, as you mentioned, especially because my sections are acetone-fixed and it could rehydrate the sections or worse, generate ice crystals.
Nicole, you're right and I always put the tissue at -20°C 30 min before cutting. I tried also PFA for fixation or before freezing (PFA fixation and sucrose gradient before inclusion in OCT) but unfortunately some of my antibodies don't work anymore...
I cut frozen brain tissue, and I notice that when I cut part of the brain, put it back in the -80, then go back to cut the next day, the first several slices from the tissue do not hold together. They are much more fragile and break apart. I suspect that the temperature fluctuations on that surface contribute to poor sectioning in subsequent sessions. Because of this, I section the entire brain region of interest in one session so I don't lose valuable material. Depending on how much/what area of the kidney you study, you could just sacrifice the top layers of tissue on subsequent sectioning days, and then the remainder of the tissue should section ok. With this approach, you don't have to section the entire kidney in 1 sitting, you will just lose some tissue each time you go back to section it.
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