FROZEN SECTIONS 



132 FROZEN-DEHYDRATION METHOD 



eosin (5 sec), cleared in carbol xylol, 

 blotted with filter paper and mounted 

 in balsam. Proescher, F., Proc. Soc. 

 Exp. Biol, and Med., 1933, 31, 79-81 

 recommends pinacyanol as giving ex- 

 cellent color contrasts. Perhaps the 

 simplest method advised by the Bens- 

 leys (p. 138) is to stain the sections in 

 Goodpasture's Acid Polychrome Meth- 

 ylene Blue (which see) 1 min. or longer, 

 wash and mount in aq. dest. This 

 colors nuclei dark purple and connective 

 tissue bright rose red. But methylene 

 blue is less permanent than hema- 

 toxylin. Adamstone and Taylor (Stain 

 Techn. 1948, 23, 109-116) have devel- 

 oped a modified technique useful for 

 histochemical and routine studies. 



For reticular and collagenic fibers in 

 frozen sections proceed as follows 

 (Krajian, A. A., Arch. Path., 1933, 16, 

 376^378) : After fixation in 10% for- 

 malin, cut sections 5-10 microns and 

 wash in aq. dest. Then 10% aq. 

 NH«OH at 60°C., 15 min. Wash in 3 

 changes aq. dest. and place in 0.3% 

 KMn04 for 5 min. Rinse in aq. dest., 

 decolorize in 1.5% oxalic acid until 

 brown color has entirely disappeared. 

 Wash 4-5 times in aq. dest. and soak 

 in 5% AgNOj at 60°C. for 1 hr. Wash 

 twice in aq. dest. Transfer to ammoni- 

 acal silver sol. (to make add 6 drops 10% 

 NaOH to 8 cc. 10% AgNO,. Then add 

 freshly prepared 10% NH4OH drop by 

 drop until almost entirely clear. Dilute 

 to 28 cc. with aq. dest.) 16 min. at 60°C. 

 Wash 3 times quickly in aq. dest. 

 Change to 30 cc. formaldehyde + 70 cc. 

 aq. dest. 1-3 min. at 60 °C. Wash in 

 tap water. Mount on slide. Dehydrate 

 with a little absolute alcohol and blot 

 into position. Dehydrate more, blot, 

 3 changes equal parts anilin oil and xylol, 

 xylol, balsam. Reticular fibers jet 

 black, collagenic ones dark brown. 



For serial sections of brain (Marshall, 

 W. H., Stain Tech., 1940, 15, 133-138) 

 fix slices 24 hours or longer in 10 or 

 15% formalin and then treat them with 

 a 20-30% alcohol or in 15% formalin in 

 20% alcohol. The object of the alcohol 

 treatment is to avoid formation of hard 

 and brittle ice crystals which fracture 

 the sections as they are made. Cut 

 tissue into blocks about 1.0 cm. thick. 

 Place on a CO2 ice freezing disc which 

 has been covered by a piece of wet blot- 

 ting paper. (In our laboratory we use a 

 regular COj gas freezing disc which has 

 been adapted to a precision sliding 

 microtome.) Freeze the block of tissue 

 slowly throughout. The proper degree 

 of freezing depends on the thickness of 

 the sections to be cut. Marshall recom- 

 mends a paraffin knife, 20-30° angle with 



block, knife set in a line perpendicular 

 to the direction of motion. Remove cut 

 sections by a camel's hair brush to 50% 

 alcohol and keep them in serial order. 

 Mount sections serially on slides coated 

 with Albumen-Glycerin. Smooth out 

 wrinkles and flatten sections by gentle 

 pressure with blotting paper moistened 

 with 50% alcohol. Remove slides to a 

 38 "C. oven for 4-6 hrs. when they are 

 ready for staining. (In some cases it 

 may be inadvisable to press the sections 

 flat upon the slide. Thin sections re- 

 quire less drying than thick ones. In 

 any case until one has gained confidence 

 in the use of the technique, the sections 

 should be observed at intervals in the 

 38 °C. oven. At the least sign of exces- 

 sive drying (whitening of parts of the 

 section) the sections shoula at once be 

 removed to the stain.) The Cresyl 

 Violet method of Tress and Tress is 

 recommended. 

 Frozen-Dehydration Method for histologi- 

 cal fixation — Written by Normand L. 

 Hoerr, Department of Anatomy, West- 

 ern Reserve University School of Medi- 

 cine, Cleveland, Ohio. November 28, 

 1951 — This Method has distinct ad- 

 vantages over the usual methods of 

 preparation of tissues for microscopy, 

 particularly for histochemical studies 

 and for microincineration. Tissues are 

 frozen as rapidly as possible and then 

 maintained below the freezing point 

 while being dried in vacuo. The 

 method changes tissues little or not at 

 all chemically, and physically only by 

 the removal of water. If the freezing 

 of a block of tissue can be effected 

 rapidly enough the ice crystals which 

 form during the freezing out of free 

 water will be below microscopic size. 

 The constituents of the tissue are then 

 not displaced to an extent appreciable 

 by microscopic examination. To pre- 

 vent displacement of any constituent 

 of the tissue after freezing, it is neces- 

 sary that the dehydration be carried 

 out below the eutectic point of the 

 tissue. There is no way of knowing 

 what the eutectic point of such a com- 

 plex mixture of substances as obtains 

 in animal tissues may be but, since in 

 tertiary and quaternary systems the 

 eutectic point is usually lower than the 

 eutectic points of mixtures of any two 

 of the substances, it is reasonable to 

 assume that the absolute depression of 

 the freezing point in tissues may be 

 well below — 54.9°C. (the eutectic 

 point of aqueous calcium chloride solu- 

 tion). From our experience, we would 

 say that better cytologic appearance 

 of the tissues — i.e., an appearance com- 

 paring most favorably with the appear- 



