I'm not familiar with brain IHC, but I'm experienced in fixing (with PFA), crioprotecting with sucrose solutions and embedding in OCT some other tissues.I've reacently tried a whole mouse brain for the first time and I'm having difficulties.
I understand how important is vascular perfusion for doing Inmunohistochemistry in the brain, but I currently don't have access to it, so I carefully retrieved a mouse brain, washed it in chilled PBS and halved it coronally. I submerged the posterior part (medulla, pons, cerebellum and the posterior parts of the cortex) in PFA 4% overnight on an orbital shaker.
Next day I washed it, submerged it in 10% sucrose for about 4 hours and then 30% sucrose overnight. After that I embedded it on OCT on a mold and started making cuts in the cryostat.
I started cuttting coronal sections from the front, noticed the cortex had receded somewhat, or maybe it was the midbrain going forward. Anyway i carried on and tried to get some clean 10um slices to check in the microscope with the aid of the Praxinos book. I was safely far from my target yet. I'm looking at getting slices of the cochlear nucleus of the medulla, I am hoping being these nuclei so superficial I might get the chance to have them well preserved even though I'm not doing vascular perfusion.
My problem is whenever I stick the brain slices to the superfrost slides. Looks like they stick allright but in the middle, where the tissue hangs over the glass like a bubble, and produces distortions on most of the brain slice. I wasn't able to fix this yet. I've tried using warmer temperatures (-15 to -20ºC) unsucessfully and I'm not sure what to try next.
Hi there,
Are you using Superfrost or Superfrost Plus slides? Fixed & frozen tissue will not stick to the normal Superfrost slides
Hello Laura, I think they must be superfost plus. I've used the same ones to do IHC on eyes and inner ear before without problem.
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