Rhesus Macaque Brain Fixation and Cryopreservation
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Corresponding Organization :
Other organizations : University of Fribourg, California Institute of Behavioral Neurosciences and Psychology (United States), University of California, Davis
Protocol cited in 8 other protocols
Variable analysis
- Intravenous injection of sodium pentobarbital (50 mg/kg i.v., Fatal-Plus, Vortech Pharmaceuticals, Dearborn, MI)
- Perfusion transcardially with ice cold 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 minutes at a rate of 250 ml/min
- Perfusion with ice cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 minutes at a rate of 250 ml/minute, then continued for another 50 minutes at a rate of 100 ml/min
- Postfixation of the brains in the same fixative at 4°C under constant, gentle agitation for 6 hours
- Immersion of the brains in a cryoprotective solution made of 10% glycerol and 2% dimethyl sulfoxide (DMSO; Fisher Scientific, Waltham, MA) in 0.1M phosphate buffer for 24 hours at 4°C
- Immersion of the brains in 20% glycerol and 2% DMSO in 0.1 M phosphate buffer at 4°C for 72 hours
- Cutting the brains into three blocks in the coronal plane using a histological blade
- Flash freezing the blocks in isopentane (2-methyl-butane, Fisher Scientific, Waltham, MA) cooled in a 100% ethanol dry ice bath
- The two adult rhesus macaques (one 13-year-old male and one 4.5-year-old female)
- The use of 0.1 M phosphate buffer (pH 7.4) throughout the perfusion and cryoprotection steps
- The use of constant, gentle agitation during the postfixation step
- The use of 4°C temperature during the cryoprotection steps
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