Good day,
We recently ran an experiment for c-fos staining within the amygdala.
90 minutes after the behavior, the animals have been transcardially perfused with PFA and the brains have been put in PBS-sucrose after 2h.
Our tech forgot the brains at room temperature within the sucrose solution since 2 weeks, without being sliced.
Do you think that this will have an impact on the final staining ?
Thanks for your answer !
Hi Noemie,
In my experience, long-term storage of whole brains in 30% sucrose is perfectly fine, but it's usually done at 4°C. If the perfusion was good enough and nothing has grown into the sucrose solution, it's probably OK. I'm just a little bit worried with your post-fixation time: 2h seems quite short, so if some brains were badly perfused, leaving them at RT for so long might have an impact...
You'll have to test it, but honestly, I'm pretty sure it's fine!
Hi Noemie Mermet-Joret ,
Well, I agree with Vincent Simon on the shortness of the period and that some brains might not just be perfused to the extent that they are fixed as some biomarkers like c-fos can have a significant drop in detection with staining. But overall, room temperature will definitely have an impact considering that the brains have been left for 2 weeks!
Though leaving whole brains in PBS or sucrose for a couple of weeks may not have such impact for other goals (sectioning for cannulation or electrode placement as histological verification), this would be problematic for c-fos or inflammatory biomarkers.
I would section one and stain a few slices to see if they are suitable or not.
Thank you all for your answers. I sliced some of them, and did the staining. I will soon go to picture them with confocal, when I will have access to the lab during this crazy situation.
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