66 Freezing Method (chap, fi) 



excess water and place the slides in a covered dish over formalin. This 

 converts the gelatine into an irreversible gel, thereby holding the sec- 

 tions in place. After 30 minutes over the formalin, ^vash in running 

 water, 10 minutes, and proceed to stain. 



Gelatine Embedding 



Culling (1957) Method 



Some tissues tend to fall apart when sectioned, and it is necessary to 

 embed them in some medium such as gelatine. 



1. After fixation, wash. 



2. Place in 10% gelatine in 1% phenol aqueous, 37 °C: 24 hours. 



3. Transfer to 20% gelatine-phenol, 37°C: 12 hours. 



4. Embed in 20% gelatin using a mold as for paraffin. 



5. Allow to set, preferably in cold. Trim off excess gelatine close to tis- 

 use. Leave as little as possible on under surface; the gelatine tends to 

 inhibit freezing. The tissue block should be small with a minimum 

 of gelatine between the tissue and the freezing stage. 



6. Before freezing, immerse trimmed block in 10% formalin to harden: 

 24 hours. 



7. Section as usual on the freezing microtome. 



8. Float sections on cold water and transfer to albumenized slides. 

 Drain, and warm slightly to coagulate albumen. Place in warm wa- 

 ter to remo^ e gelatine and proceed to stain. 



Pearse (1953) Method 



1. Fix in cold formalin (15%) at 4°C if it is desired to preserve en- 

 zymes: 10-16 hours. 



2. Wash, running water: 30 minutes. 



3. Infiltrate with gelatine, 37°C: 1 hour. 



gelatine 15.0 gra. 



glycerol 15.0 ml. 



distilled water 70.0 ml. 



small crystal of thymol. 



4. Cool and harden in formalin (40%o), 17-22°C: 1 hour. \Vash. 



5. Store at 4°C or below until sectioned. 



See Albrecht (1956) and Thompson (1957) for staining in quantity. 



