T.S. Fat 



FROZEN SECTIONS 



161 



block is then left to harden in 2% formal- 

 dehj'de for 24 hours. 



The hardened block is now taken and 

 placed on the freezing stage of a micro- 

 tome, preferably a slider of the type shown 

 in Fig. 55. The block is carefully oriented 

 on the stage and chilled with the aid of a 

 stream of carbon dioxide until sections 

 may be taken from it. If the block has 

 been properly made, and is cooled to the 

 correct extent, it behaves almost exactly 

 as though it were a nitrocellulose block. 



Each section, as it is cut, is removed 

 from the knife with a brush, and the sec- 

 tions are accumulated in 50% alcohol. 

 Each section is then taken individually in 

 a pair of forceps, dipped for a moment in 

 5% gelatin, and then placed in the center 

 of a clean slide. These sections are flexible 

 and may be flattened to the slide without 

 difficulty. As soon as the surplus fluid has 

 been drained off, the shde is placed in a 

 coplin jar at the bottom of which are a few 

 millihters of 40% formaldehyde. Exposure 

 to formaldehyde for about an hour will fix 

 the section firmly; the process may be ac- 

 celerated in an oven at a temperature of 

 37°C. The slides, after hardening in 

 formaldehyde vapor, may be stored in 2 % 

 formaldehyde. 



The next problem is that of staining and 

 mounting. In the writer's opinion the best 

 stain for the demonstration of fat in sec- 

 tions of this type is that of Lillie 1945 

 (Chapter 21 DS 22.4). This method re- 

 quires a saturated solution of oil blue in 

 60% isopropanol, a 0.1% solution of 

 Bismark brown R in water and some 5% 

 acetic acid. The slides to be stained are 

 removed from 2% formaldehyde, rinsed 



briefly, and tiansferred to the blue solu- 

 tion where they should remain from five 

 to ten minutes, or until examination under 

 the low power of the microscope shows 

 that the individual fat granules have ab- 

 sorbed the blue dye. They are then briefly 

 rinsed in water and transferred to Bismark 

 brown solution for one to two minutes. 

 When they are removed from this solution 

 they will appear to be a muddy brown, 

 and should then be rinsed in 5% acetic 

 acid until examination under the micro- 

 scope shows the fat granules to be brilliant 

 blue while the supi)orting connective tis- 

 sues are stained brown. The slide should 

 then be rinsed in water to remove the 

 acetic acid and return to 2 % formaldehyde 

 to await mounting. 



It is obvious that slides of this nature 

 cannot be mounted in a resinous medium, 

 for the necessary dehydrating and de- 

 alcoholizing agents would remove the fat 

 from the sections. It is therefore necessary 

 that they be either mounted in glycerol 

 jelly in the manner described in Chapter 

 5, or (in the author's opinion this is better) 

 in one of the gum media described in 

 Chapter 4. The author has naturally a 

 preference for his own formula (Chapter 

 26, M 13.1 Gray and Wess 1950). The sec- 

 tions can be mounted permanently in this 

 media by removing them from 2% form- 

 aldehyde, rinsing them briefly in water, 

 draining them, placing a drop of the 

 mounting medium on top of each section, 

 and applying a coverslip. The slides Avill 

 be hard enough to handle in less than an 

 hour, and will be as nearly permanent as 

 anv section of fatty material can be. 



