Double E/nhc(l(li)tg 83 



Goland et al. (1954) INfethod 



(ioland and coworkers follow carbowax infiltration by: 



xylene, 61°C: 10 minutes, 

 paraffin, 61 °C: 30 minutes, 

 embed. 



Chill in refrigerator, do not apply ice directly. This overcomes some 

 of the disadvantages of carbowax and produces less shrinkage and dis- 

 tortion than a regular paraffin method. After sectioning, the tissues may 

 be handled as is customary for paraffin sections. 



Either albumen or Giovacchini's fluid smeared on slides have been 

 successful methods, but it has been easier to moiuit sections out of a 

 water bath (of floating solution, Jones et al., 1959) on to gelatine 

 smeared slides than by laying the sections directly on floating solution 

 on slide. Albumen slides moinit readily either way. The razor-blade 

 method is tricky, but works. 



Double Embedding 



Fragile, small and hard objects often crumble when processed in the 

 paraffin method, and some difficulties can be eliminated by substitiuing 

 a double embedding technic. 



1 . Fix and wash tissues as usual. 



2. Dehydrate in 50%, 70% and 90% ethyl alcohol: 2 hours each. 



3. Absolute ethyl alcohol: 2-16 hoins. 



4. Methyl-benzoate-celloidin solution (see below): 24 hours. Pour off 

 and replace with fresh solution: 48 hours. If tissue is not clear, repeat 

 for 72 hours. 



5. Pure benzene, 3 changes: 4 hours, 8 hours, and 12 hours. 



6. Mixture of equal parts of paraffin and benzene, in embedding oven: 

 1 hour. 



7. Paraffin, 2 changes: ^ to 6 hours, depending upon thickness and 

 nature of tissue (3 hours for 5 mm. thickness). 



8. Embed and proceed as with ordinary paraffin sections. 



Methyl-henzoate-celloidiyi Solution 



Add 1 gram air-dried celloidin flakes to 100.0 ml. methyl benzoate. 

 Shake well and allow bottle to stand upright for an hour or longer. 



