I am going to do in situ hybridization for adult mouse brain and spinal cord. I will post-fox the tissues in 4% PFA ON and then embed in paraffin. Should I also perfuse the mice before with PFA? I know that perfusion preserves the tissue to the greatest extent, but is it necessary in this case?
Hi Iddo;
from my experiences with mouse brain immuno-fl.-staining and IHC, perfusion with PBS followed by 2nd perfusion with PFA gives high quality of tissue, however I don't know how it will work for ISH.
From my experiences with zebrafish ISH I can recommend to store tissue in methanol at -20 prior to ISH.
Probably its worth trying a mixture of both (PFA perf.and PFA storage vs storage in methanol) !?
Best,
Michael
Perfuse the animal with PBS (20 ml) first, and then perfume with 4% PFA(20 ml). We also do a 2 hour post fix in 4% PFA. It works for both ISH and IF in brain and spinal cord tissue in our lab.
Debarghya Dutta Banik are these amounts (20ml PBS and 20ml PFA) good for an adult mouse?