X-Message-Number: 11600
Date: Fri, 23 Apr 1999 05:26:54 -0700 (PDT)
From: Doug Skrecky <>
Subject: cryoprotection of fixed monkey brains

  Rosene DL.  Roy NJ.  Davis BJ.
  A cryoprotection method that facilitates cutting frozen sections of whole
  monkey brains for histological and histochemical processing
  without freezing artifact.
  Journal of Histochemistry & Cytochemistry.  34(10):1301-15, 1986 Oct.
  Cutting frozen sections of large (greater than 60 cc) blocks of monkey
  brain using the conventional procedures of infiltration with
  30% sucrose as a cryoprotectant before freezing with
  pulverized dry ice often produces unacceptable levels of freezing artifact
  (FA) caused by displacement of tissue by ice crystals. Experiments
  investigating FA utilized perfusion-fixed brains from 46
  monkeys and spanned combinations of cryoprotectants
  (glycerol, sucrose), freezing methods (dry ice or -75
  degrees C isopentane), and fixatives (10% formalin, Karnovsky's or Timm's).
  The effects were evaluated by rating of FA severity in frozen sections of
  whole monkey brains. Minor FA appears as enlarged
  capillaries, more serious FA as large vacuoles, and both first appear midway
  between the periphery and center of the block. Stronger fixatives increased
  the severity of freezing artifact. The best method for eliminating FA was
  graded infiltration with up to 20% glycerol and 2% DMSO (in
  buffer or fixative), followed by rapid freezing in -75 degrees C isopentane.
  Although using a glycerol-DMSO infiltration before
  conventional freezing with pulverized dry ice or using conventional sucrose
  infiltration before freezing in isopentane gave better results than sucrose
  infiltration and dry-ice freezing, only the combination of
  glycerol-DMSO infiltration and freezing in isopentane
  produced consistently excellent results and virtually eliminated freezing
  artifact. To determine the effect of freezing with dry ice or isopentane on
  the rate of cooling in large blocks of CNS tissue, thermocouples were
  embedded in an 80-cc block of albumin-gelatin and frozen with the two
  methods. The rate of cooling (-3.5 degrees C/min) was twice as fast using

  Additional note by poster:

    Sucrose can penetrate only fixed tissue, which has larger pores in
  cell membranes.

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