METHODS IN MAMMALIAN IMMUNOGENETICS 371 



ment. In many instances, such reactions "fix" complement if it is present, even 

 though the complement may not be necessary for the reaction to occur. After this 

 phase of the test, in which the antigen-antibody reactions occur, a second indicator 

 system is added in order to determine whether complement has been fixed. The 

 indicator system is usually sheep cells which have been sensitized with rabbit-anti- 

 sheep antibody. If complement is present, the cells will hemolyze; if it has been fixed 

 by a preceding antigen-antibody reaction system, it will not be available to the hemo- 

 lytic system and the cells will fail to lyse. The sensitized sheep cells, therefore, provide 

 a measure of whether or not an antigen-antibody reaction has occurred in the test 

 system. 



Techniques of complement fixation are highly sensitive, and are subject to modifi- 

 cation by many external factors. Careful controls must be run ; the proper amount of 

 complement must be added; the indicator cells must be properly sensitized, and so on. 

 Descriptions of complement fixation procedures are to be found in Kabat and Mayer 673 

 and Boyd. 122 In general, although this system is a useful one in serology, it has found 

 relatively little application in immunogenetics, particularly of mammalian materials. 



TISSUE TRANSPLANTATION 



Consideration of tissue transplantation will be limited to studies of normal rather 

 than neoplastic tissues and to only a small selection from this active area. The method- 

 ology of tumor-transplantation is considered elsewhere in this volume. 



Skin grafting. — The standard method for skin grafting, especially in mice, was 

 developed by Medawar and his group. 98a The following technique, which we use in our 

 laboratory, is essentially Medawar's. 



Donor mice are usually killed by the intraperitoneal injection of 0.1 ml. of Nem- 

 butal sodium, 50 mg. per ml., which is commercially available. Alternatively, if not 

 too many grafts are to be removed, the donor mice may be anesthetized by the injection 

 of 0.1 ml. per 10 g. body weight of a 1/10 dilution of the above Nembutal preparation. 

 Different lines of mice vary somewhat in their sensitivity to the anesthetic, and the first 

 mice to be injected should be carefully observed, with the idea that slightly less or more 

 anesthetic may be desirable for the line in use. After the donor mouse has recovered 

 from anesthesia, the sites from which the grafts are taken may be left open; they will 

 heal rapidly. 



If the donors have been killed they can be pinned out on a board; if they have not 

 been killed they can be tied or taped down. The back of the animal, from the ears to 

 the tail, is clipped closely ; we use an Oster small-animal clipper, model 2, with a no. 40 

 head. The surface of the clipped skin is then swabbed thoroughly with Zephiran 

 (1/1000 in 50 per cent alcohol). 



An area of the skin of the donor is elevated in a small tent, with a small, very fine 

 pointed forceps, the tips of which have been curved toward each other to meet at a 

 single sharp point. As the skin is held under tension, it is cut from beneath with a 



