[Histonet] Rat brain in 70% alcohol for cryotomy

From:"Gayle Callis"


You wrote 

We received a few whole rat brains in 70% EtOH after perfused with 10% BF. Now they changed mind want go for frozen. The want Cresyl Violet stain. Is it doable? How        

The problem is that 70% alcohol acts as antifreeze, and unless you remove the alcohol, the brain will not freeze properly.  The alcohol can be replaced with a sucrose cryoprotection, needed even after NBF fixaiton for proper snap freezing. 
You could sucrose cryoprotect the brains in order to prepare them for  snap freezing and cryotomy, but it may take several days using an increasing concentration/sucrose gradient to replace the alcohol with sucrose solution . You can start  in 15% sucrose, 20% sucrose and then 30%    sucrose.  I am not sure of timing for a whole rat brain, but overnight  in each change at 4C may do the job. 

It may be easier to just process into paraffin and do the cresyl echt  violet on paraffin sections instead, unless they are asking for thick sections?   We do cresyl violet staining on paraffin sections instead of frozen sections, but the stain can be done on frozen sections.  Histonet Archives has discussed sucrose gradients, the problem you presented and also doing cresyl echt violet on frozen sections - you may want to do a search to find out what others have discussed.   

Good luck,
Gayle Callis MT,HT,HTL(ASCP)  
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