I am currently working in Sepsis brain model in mice. I have to use the mouse brain for immunohistochemistry and RNA synthesis purpose. As we know for immunohistochemistry purpose we perfuse mouse brain using fixative such as 4% PFA through transcardial route and for RNA extration we should avoid the fixation step.
At this ground I have to use single brain for both purpose. I tried using DNA extraction kit from fixed tissue section, but the results were not that reliable.
So at this time I am planning not to fix the brain with fixative through transcardial perfusion, rather first divide the brain into 2 hemisphere, take 1 for RNA extraction purpose and next for immunohistochemistry purpose. for Immunohisto purpose I will post fix the hemisphere using 4% PFA and proceed accordingly.
But I have strong doubt on this purpose, So if you have any better methods please share with me. Thank you.
Why do you have doubts ? This is the logical way to proceed, and really your only option if you want to do both RNA and IHC work. Mouse brain fixes perfectly well by simple immersion in formaldehyde. Just make sure your formaldehyde solution is well-prepared (fresh, and without precipitates), and that you have at least 10 volumes of solution per volume of tissue.
Prepare a solution of 4% paraformaldehyde (PFA) in phosphate buffer (PBS). Cinar et al. Acta Histochemica 2006 v. 108(6); 487–496 compared immunohistochemistry for several proteins after fixation samples of different mouse organs (brain, kidney, liver and heart in acetone, formaldehyde (FA) and 4% PFA with and without perfusion - PFA fixation was better than AC and FA. They performed IHC on frozen sections, but the same is true for tissues processed to paraffin wax blocks. Jean-Martin gave you a good advice that you need to use at least 10 times volume of fixative per volume of tissue, - and preferably on orbital shaker. As far as I know, some researchers opt for 24 hour fixation, but check the literature. Good luck with your experiments.
Thank you very much to Jean-Martin Lapointe and Maria Jeziorska for your nice advice
1.transcardial perfusion with PBS
2.seperate two hemispheres
3.Fix a hemisphere with paraformaldehyde 4% in PBS for 24 hr
Dear Naomi
For RNA extraction perfusion with PBS is fine. I have routinely done it and got the good results (RNA purification index ~2.0). So I have no alternatives to follow above suggestion and that I had planned too (already). Tomorrow is the sampling day !!
Cheers!!
Dear Bhakta,
Please can you tell me if this experiment worked because I woude like to use the same protocol for our mouse brain experiment- Thanks
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