CARBOWAX EMBEDDING 



59 



CAREY'S 



Maryland. February 9, 1951 — Carbo- 

 wax (Carbide and Carbon Chemical 

 Division, 30 East 42nd Street, New- 

 York 17, N. Y.), a water soluble wax, 

 is an excellent embedding medium for 

 tissues. It circumvents the long de- 

 hydration process necessary for em- 

 bedding in paraffin or celloidin and yet 

 sections prepared by this method show 

 excellent preservation of cytological de- 

 tail. Sections of Carbowax embedded 

 tissue are easy to cut at from 1 to 10 ^i 

 in thickness which makes this embed- 

 ding method well suited both for the 

 study of minute cytological details and 

 for routine histological examinations. 

 In contrast to paraffin and celloidin 

 embedded tissues, lipids can be studied 

 in sections of the same tissue block 

 (Firminger, H. I., Stain Tech., 1950, 

 25, 121-123). Any type of fixation may 

 be used and almost all staining pro- 

 cedures can be performed on Carbowax 

 sections with the exception of osmic 

 acid. Staining with osmic acid is 

 better after the conventional methods. 



Carbowax is not miscible with fats 

 and penetrates adipose tissue, brain or 

 spinal cord only very slowly. Such 

 tissues can sometimes be embedded 

 after long periods of infiltration in the 

 oven with resultant distortion or by 

 previous removal of lipid with fat sol- 

 vents. Other disadvantages are the 

 difficulty in mounting ribbons on the 

 slide because of the solubuility of the 

 Carbowax in the aqueous bath used for 

 floating the sections, and the difficulty 

 of making sections stick on the slide 

 during the staining procedure. Blank 

 et al. (Blank, H. and McCarthy, P. L., 

 J. Lab. and Clin. Med., 1950, 36, 776- 

 781) recommend placing sections on an 

 aqueous bath containing gelatin and 

 potassium dichromate, floating sec- 

 tions onto a clean slide and drying 

 thoroughly. An alternate procedure 

 which avoids exposure to chromates 

 (undesirable for certain staining pro- 

 cedures) is to cover the sections with 

 thin colloidin prior to staining. Blank 

 has also suggested mounting ribbons 

 directly on a slide wet with the gelatin- 

 dichromate solution. 



The solution recommended by Blank 

 et al. for floating and affixing sections 

 to slides is made by dissolving 0.2 gm. 

 of potassium dichromate and 0.2 gm. of 

 gelatin in 1000 cc. aq. dest. The mix- 

 ture is boiled in daylight for five 

 minutes, cooled and filtered. 



Embedding in Carbowax ordinarily 

 employs a mixture of Carbowax "4000" 

 and Carbowax "1500". The exact pro- 

 portions of the components must be 

 varied to suit climatic conditions. 



Pure Carbowax "4000" may be neces- 

 sary in hot climates. In cooler climates 

 one can use a mixture composed of 85 

 gms. of Carbowax "4000" and 15 gms. 

 Carbowax "1500". For use, the Carbo- 

 waxes are combined in the proper pro- 

 portions, heated to 175°C. for 30 seconds 

 and placed in an oven at 56°C. This 

 solution should be kept in the oven. If 

 the temperature of the oven should drop 

 and the Carbowax mixture solidifies, 

 it requires reheating above 56°C. to 

 remelt it. To embed: 



1. Place fixed tissue in small Stender 

 dish containing the above Carbowax 

 mixture in oven at 56°C. 1-3 hrs. 



2. Agitate dish and contents every 10 

 min. to aid penetration. All tissues, 

 including lung tissue, should sink before 

 further embedding. 



3. After sufficient infiltration remove 

 tissue and place in fresh mixture in 

 another Stender dish. 



4. Cover dish and place in icebox to 

 harden at 5°C. to avoid crystal forma- 

 tion. 



5. Trim block with hot knife. 



6. Block on hot object holder; then 

 cool object holder with ice, taking care 

 that water or ice do not come in con- 

 tact with block. 



7. Cut sections (1 to 10 n). If satis- 

 factory ribbons are not obtained the 

 procedure is not working properly. 



8. (a) Float sections on a water bath 

 at room temperature to which 2 drops 

 of a detergent or the first ribbons of 

 Carbowax have been added to prevent 

 "explosion" of the sections by the sur- 

 face tension. Stain sections before 

 mounting on slide; or, float sections 

 onto the slide from the water bath, 

 cover carefully with a few drops of a 

 mixture of equal parts of absolute al- 

 cohol and ether. Pour off excess and 

 cover with thin collodion; harden in 

 70% ale. or water. Stain. 



(b) Or, place sections on a bath con- 

 taining 0.02 gm. % gelatin and 0.02 gm. 

 % potassium dichromate. Float onto 

 clean slide, permit to drj^ thoroughly 

 and stain. 

 Carey's method for motor end plates is an 

 adaptation for his study of their ameboid 

 motion (Carey, E. J., Anat. Rec, 1941, 

 81, 393-413) of Wilkinson's (H. J., Med. 

 J. Austral., 1929, 2, 768-793). Modifica- 

 tion of Ranvier's gold chloride technique. 

 — Written by the late Dr. E. J. Carey, 

 Dept. of Anatomy, Marquette Uni- 

 versity School of Medicine, Milwaukee, 

 Wis. 



1. Remove any muscle from rat or 

 chameleon from its origin to insertion 

 while the animal is under ether ornemo- 

 butal anesthesia. Using a very sharp 



