[Histonet] hand processing of rat brain revisited

From:Lori Roberts

Hi there,
  I need some advice on a hand processing schedule for rat brains.  Because I am the only person at my company with histology experience (limited to some paraffin embedding and sectioning I did of embryonic tissues in grad school) I have been asked to set up a histology lab from scratch on short notice.  We are trying to replicate an experiment that was done at a contract lab, and we need results before the end of the year.  The contract lab provided us with all of their protocols, but of course they use automated processing, with vacuum at every step.  I am processing cassettes like I did in school--in a polypropylene tri-pour beaker that I burned holes into inside another beaker with a stirbar in the outer beaker (no vacuum).  The paraffin steps are carried out in a vacuum oven at 58C/-15 mm Hg vacuum.  When I tried to process some practice brains, the sectioning was terrible--brittle tissue and jammed up sections.  One problem is that I used too small a volume of
 fix--only 20 mL before reading in Frieda Carson's Histotechnology text that I should use at least 20X volume (like I said, my only experience was with tiny tissues where volume was never an issue).  I am fixing some new brains in 60 mL NBF per brain.  But before processing these I thought I'd get some professional opinions about whether the schedules I have tried are appropriate for rat brain.  
  Here are the two schedules I tried:
  Contract lab's schedule (they use this with vacuum at every step, I have vacuum only for paraffin):
  Whole rat brain fixed 10% NBF (20 mL) 36 hours
  Trimmed into 3 mm slices of forebrain and midbrain, placed in cassettes and stored O/N in formalin
  30 min 70% EtOH
  30 min 80% EtOH
  30 min 95% EtOH times two
  30 min 100% EtOH times three
  45 min xylenes times two 
  40 min ParaPlast Xtra 58C/-15 mm Hg times two
  80 min ParaPlast Xtra 58C/-15 mm Hg
  After these didn't section well, I took some remaining pieces of the above fixed brains to try the schedule I used to use in graduate school for embryonic tissues:
  Leftover brain pieces from first try stored in same 20 mL NBF vial as above 7 days
  Trimmed into 3 mm slices, placed in cassettes and immediately processed
  45 min 70% EtOH
  45 min 95% EtOH
  60 min 100% EtOH
  45 min xylenes times three
  60 min ParaPlast Xtra 58C/-15 mm Hg times two
  I have ordered an inexpensive Nalgene vacuum chamber so I will soon be able to apply vacuum at the earlier dehydration steps.  My next question concerns the vacuum pressure.  I have seen values in the Histonet archives ranging from -15 mm Hg to 40-200 mm Hg to 15 inches Hg.  The gauge on my vacuum oven only goes down to -30 mm Hg.  How much vacuum should I be using?
  Sorry for the long message, but I know the devil's in the details.
  Thank you,
  Lori Roberts

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