My lab is looking to perform IHC staining on the brains of several dozen mice, and we have a need to store the perfused brains for up to a month before we have access to paraffin embedding. Can anyone recommend a protocol that can prevent damage to these brains for that period of time?
Depends on your end point (i.e. stain target Immunohistochemical or Nissl), meaning you don't want to over fix your tissue if your target is immunological. However if your tissue doesn't need to have antibody/antigen cross reactivity to visualize your target of interest, such as cresyl violet staining, it may be possible to store in buffered formalin or 2-4% PFA (RT or 4C). On the other hand you could perfuse and fix as normal and after which store in 0.1MPBS at 4C until you can begin the dehydration and embedding process.
Thank you Jeffrey P Cheng, we will be performing immunohistochemical analysis on these slides. Is the second option you mentioned, storing them in 0.1M PBS at 4C until dehydration, appropriate to store these tissues for ~1 month before doing paraffin embedding or would it just be best to do the embedding immediately for each sample?
If you can dehydrate and embed immediately that is the best solution. I may have misunderstood the question regarding access to paraffin embedding. Once dehydrated and embedded in paraffin wax they can remain that way indefinitely. It would be the ideal way to keep brains for staining. Be sure that your antibody has been characterized with paraffin sectioning, as not every antibody works well with paraffin. Keeping your post-fixation protocol and embedding protocol consistent between batches will significantly reduce variability between batches.
I m in support of Jeffrey P Cheng first option, you can preserve your samples in a mild preservative such as 4% paraformaldehyde so as to prevent denaturation of the proteins. Because based on your statement, you don't have access to paraffin embedding for now. Thank you.
- Badges
- Science topic