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Fixation and Dehydration

1. Obtain a fresh mouse liver, and cut into pieces less than 1 mm across. Keep all of the tissue submerged under a bubble of physiological saline at all times. VIEW
2. Immerse in physiological saline solution, each piece in its own test tube. Let sit for three hours, changing to a new test tube of solution every hour. Transfer the pieces using a pipette. Make sure that each test tube has been thoroughly cleaned. VIEW
3. Transfer the pieces to test tubes with fixative and allow to sit for about 12 hours.    
4. Transfer to test tubes with treatment and allow to sit for 1 hour.    
5. Transfer to test tubes with post-fixative and allow to sit for 1 hour.    
6. Dehydrate using a series of graded ethanol washes. NOTE: Be sure to tightly seal each wash with Parafilm before placing the pieces in the wash and while the tissues are in the wash.    
6.1. Transfer all the pieces to a small beaker of 30% EtOH (190 proof) for 10 minutes and then to the following washes:
6.2. 40% of 190 proof EtOH - 10 mins
6.3. 50% of 190 proof EtOH - 10 mins
6.4. 70% of 190 proof EtOH - 10 mins
6.5. 80% of 190 proof EtOH - 10 mins
6.6. 90% of 190 proof EtOH - 10 mins
6.7. 100% of 200 proof EtOH - 10 mins
7. Immerse tissue pieces in HMDS for 3-5 minutes.    
8. Blot dry with tissue and let air dry in desiccator on Parafilm.    

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