I'm trying to slice unfixed bird brains that were preserved at -20°C in RNAlater. It seems that the tissues are not frozen properly when I'm trying to cut them in a cryostat, probably because of the RNAlater (the tissue was frozen in tissue-tek on dry ice before). The tissue unravels inside the mold when I try to slice it. I want to perform an in situ hybridization on these brains. Anybody have a solution?
Tissue stored in RNAlater is not fixed. There will be little tissue architectural integrity left even stored at -20 C. There will be major protein disruption (RNAlater is designed to lyse cells) and as a consequence any RNA localisation in tissues stored in RNAlater will be eroneous. I suggest perfusion fixation or immediate flash freezing of the brains to be studied. Cryotomy of the brains you have already harvested and stored in RNA later may improve slightly by cutting at a lower temperature, however I have had little success with gut even down to -50 C. Good luck
Hi,
I have a feeling the tissue is not fixed properly. How did you preserve it in RNA later? Did you allow for the RNAlater to penetrate the tissue long enough before putting into the freezer? I assume that a bird brain is relatively large and you may need to check how long you have to keep the brain in the RNAlater for proper fixing.
Cheers
No, the tissue is not fixed, and RNAlater does not really fix it, it's a RNA protection agent. That is, I followed the recommendations wich are to keep the tissue at 4°C overnight to let the solution penetrate, and then store it at -20°C. I also cut the brains in half to make sur the solution penetrates the whole tissue.
Hi,
I guess it stabilises the structure of the tissue and fixing is probably the wrong word to use. Irrespective, in our experience the tissue gets hard after RNAlater treatment. How large is your bird brain and how much solution did you use? The recommended protocol is 0.5 g tissue +about 2.5 mL RNAlater and allow to penetrate O/N. If your brain is relatively big, it will take time for the solution to penetrate.
Hope you sort it out.
RNA later is claimed to be good enough to stabilise tissue for H&E (frozen section of human skin preserved for one week in RNAlater®). So even if subsequent in situ hybridisation does not work, there should be no problem with sectioning.
Aniko and Elizabeth, thanks for your answers. I know the main application for RNAlater is for RNA extraction, however, Aniko you are right that it is claimed that RNAlater should preserve tissue integrity for histological purposes. However I did not find any publication/protocol where they describe the use of RNAlater and cryostat sectionnig in particular.
Merci Caroline pour ta réponse. Could you give more details on how you achieve that? Should I use paraffin oil? Do you think I will be able to embed my tissue in tissue-tek (O.C.T.) after? And what is heavy agard?
I know that RNALater helps preserve RNA for subsequent extraction. Since your aim is to perform in situ hybridization, I assume that you have frozen it afterwards. How did you freeze it? Did you place it in the freezer directly? Fixing the brain in OCT compound subsequent to storing in RNALater overnight at room temperature or at 4 degree C may preserve both the tissue architecture and the integrity of RNA. It is helpful to perform cardiac perfusion with 10% buffered formalin to remove blood cells and to preserve the tissue architecture for histological analysis. I am wondering whether RNA remains intact either for extraction or for in situ hybridization following perfusion and freezing in OCT.
Yes, I have tried to cut some frozen breast tumours.
It was like trying to cut chewing gum.
The tissue is not frozen solid.
I thought it was just fatty.
It was quite similar to tissue that was placed in warm iso-pentane and then frozen.
The tissue is infiltrated with iso-pentane which freezes in liquid Nitrogen but at -20 oC the iso-pentane is liquid.
Usually iso-pentane is cooled first in liquid Nitrogen before tissue is placed in it.
Good luck.
Try the lower cryostat temperature. I had some problems with adipose tissue, -40 oC helped.
Tissue stored in RNAlater is not fixed. There will be little tissue architectural integrity left even stored at -20 C. There will be major protein disruption (RNAlater is designed to lyse cells) and as a consequence any RNA localisation in tissues stored in RNAlater will be eroneous. I suggest perfusion fixation or immediate flash freezing of the brains to be studied. Cryotomy of the brains you have already harvested and stored in RNA later may improve slightly by cutting at a lower temperature, however I have had little success with gut even down to -50 C. Good luck
I believe that while RNAlater claims to preserve tissue integrity, you still need to formalin fix for histological uses. The study that the claim comes from immersed samples in RNAlater overnight, then took what they wanted for RNA, rinsed the remaining portion in PBS, then fixed in formalin, then proceeded with histological analysis. You may want to try that instead, and it should help with the tissue hardness as well.
We tried a technique similar to Marilyn recently.
We had good results by putting the tissue in RNA later in the fridge overnight (mouse organs) in eppendorf tubes. The next day we bisected the tissues, keeping half in RNA later (stored at -80oC for RNA) and fixing the other half. We washed out the PBS with 2x washes for 1h each, then fixed in 10% neutral buffered formalin overnight and took the tissues through the normal fixation process. The tissues stained well.
I wouldn't advise freezing the tissues in RNA later before fixation as this may distort the tissue.
You always have to keep in mind that 'you can't have it all'. If the tissue is frozen in RNAlater the morphology is less preserved than by immediate formalin fixation. However, the same is true for tissue which is immediately cryopreserved (which, however, has a better morphology than those frozen in RNAlater), The RNA integrity is the otherway around. Using PAXgene Tissue STABILIZER from Qiagen may be the best compromise - at least in our hands. An additional level of complexity is added when you want to use the tissue for IHC or IF.
This may not be useful but there is user-developed protocol for formalin fixation and paraffin embedding after RNAlater treatment: "Preparation of RNAlater™ preserved tissues for histological
studies"
Also, fom product information sheet: "RNAlater does not dissolve or disrupt the structure of tissue samples, thus tissue"
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