[Histonet] Re: Freezing small pieces of skeletal muscle tissue


Sarah E. Lewis, Histotechnician at Mendell Neuromuscular Lab, Childrens 
Research Center for Gene Therapy in Columbus OH notes:

>>This is the protocol we send to outside institutions... You mentioned you 
wait until the 2-methylbutane / isopentane gets slushy... This we do not do. I 
listen for the "boiling" sound to
stop (so to speak). Your solution should be clear, not cloudy. You will start 
to see little white crystals form... This is when you put your tissue in the 
2-methylbutane suspended in liquid nitrogen. A 0.5x0.5x1.0 cm piece of muscle 
should be in for 30 sec. Tissue any smaller, cut your time down by 5 sec 
according to size. Hold tissue in cryostat or -20/-40 until tissue is dry. Wrap 
with foil and store in -80.<<

Many people cover the specimen with a thin layer of talcum powder before 
dipping it int the 2-MB. Also, it helps to dip the specimen up and down at about 1 
second intervals for at least 10 dips. - These strategies avoid the formation 
of large ice crystals ("waffle artifact") within muscle fibers. I'd suggest 
trying this with some practice muscle - from an amputated leg or a researcher's 
left-over dead rat, depending on where you are - before freezing a patient 
specimen - cut frozen sections to see if you have artifact.

2MB (like acetone) is a fire hazard. A few weeks ago I posted a note about 
using 3M's HFE-7100 -
methyl nonafluoroisobutyl ether
instead. Does anyone know about using this stuff as a non-flammable freeze 

Bob Richmond
Gastonia NC
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