ANTIGENS, HISTOCHEMICAL 



18 



ANTIGENS, HISTOCHEMICAL 



Because of the ever-present hazard of 

 "nonspecific" staining, careful controls 

 of specificity must be carried out at 

 every step. When the antigenic material 

 sought is foreign to the tissue under 

 investigation, control is relatively 

 simple since the conjugate should fail 

 to stain normal tissue sections. Im- 

 munologically specific staining can be 

 greatly diminished or altogether pre- 

 vented by pretreatment of parallel sec- 

 tions with unlabeled homologous anti- 

 body, thereby blocking the antigen. 

 However, replacement of unlabeled by 

 labeled antibody occurs and the time 

 of staining must usually be decreased 

 in order to carry out this control suc- 

 cessfully. Failure to stain the tissue 

 under investigation with heterologous 

 labeled antibody is also a suitable 

 control, although the degree of purifica- 

 tion of conjugates varies and it is diffi- 

 cult to establish that any two conju- 

 gates are strictly comparable. There 

 are so many possible variations that 

 controls appropriate to each situation 

 must be carefully planned. 



Fluorescence microscopy. Fluores- 

 cence microscopy is described in an- 

 other section. The amount of de- 

 posited antibody is quite small, and the 

 fluorescence therefore faint. Light 

 sources adequate for the study of 

 brightly fluorescent materials present 

 in high concentration are not appropri- 

 ate for use with labeled antibody. 

 Either a 10 amp. carbon arc or a high 

 pressure water cooled mercury vapor 

 arc (General Electric Co., AH -6) are 

 satisfactory. At this level of bombard- 

 ment, fluorescence of optical elements 

 becomes important. It is necessary to 

 use a quartz condenser since glass ones 

 often fluoresce in the ultraviolet beam. 

 Definition is improved at the cost of 

 intensity by the use of a cardioid 

 darkfield condenser. 



Photography. Findings revealed by 

 this method can be recorded photo- 

 graphically using 35 mm. film (either 

 high speed panchromatic film or fast 

 green-sensitive film, e.g., "Photoflure" 

 Eastman). Exposure times vary with 

 the amount of fluorescent antibody 

 deposited and the autofluorescene of 

 the background, varying at "high dry" 

 (400 X) from 3 to 10 minutes. Focus- 

 ing on ground glass is very difficult 

 because of the low degree of intensity 

 of the projected image, but can be 

 carried out by the use of a magnifying 

 lens focused on a clear area in the 

 ground glass. 



Anyone attempting to carry out these 

 procedures is advised to consult the 

 original papers to which reference has 



been made (cf. Federation Proc, 1951, 

 10, 558). 



Frozen Sections by the Method of 

 hinderstr^m-Lang and Mogensen 

 (Compt.-Rend. Trav. Lab. Carlsberg, 

 s6rie chim., 1938, 23, 27; Coons ,et al., 

 J. Exp. Med., 1951, 93, 173). The prin- 

 ciple of this method is quite simple. 

 It involves the quick-freezing of tissues 

 at low temperatures, and cutting them 

 while frozen in a cryostat maintained 

 at —20. This cold chamber should be 

 equipped with adequate lighting, an 

 insulated window through which the 

 operator can see, and gloved armholes 

 through which he can work. Sections 

 are cut on a good rotary microtome, 

 the knife of which is equipped with a 

 glass guide to keep the sections from 

 curling as they are cut. Convenient 

 improvements in the method are de- 

 scribed by Coons, et al. Tissues may 

 be quick-frozen by placing thin slices 

 on the side-walls of test tubes which 

 are then stoppered and plunged but 

 not submerged into alcohol cooled to 

 the temperature of dry ice. They can 

 then be stored in a deep freeze below 

 — 20, or put in the cold cryostat for 

 immediate sectioning. Long storage 

 results in the growth of ice crystals. 

 The tissue is mounted by putting a 

 small drop of water on a previously 

 chilled tissue holder and touching the 

 tissue to it. Within a few seconds the 

 tissue block will be tightly frozen to 

 the holder. Care must be taken to 

 avoid ice formation near the cutting 

 area. The knife blade should be set 

 at an angle of approximately 20° from 

 the plane of movement of the tissue 

 block. The glass guide should be ad- 

 justed so that its upper edge is parallel 

 to and at the height of the cutting edge 

 of the knife. Each section as cut is 

 removed with forceps from the knife 

 blade, placed gently on a cold slide, 

 and thawed by placing a finger under 

 the section. It can then be dried in 

 the air stream from a fan at room tem- 

 perature. Appreciable movement of 

 tissue components during this momen- 

 tary thawing has not been troublesome, 

 although it could be serious in some 

 situations. 



An adhesive is necessary on the slides; 

 the one in use by the authors quoted 

 has been formalinized gelatin. Acid 

 cleaned, dry glass slides are dipped in 

 0.5% gelatin, and placed on end to 

 drain and dry. When dry, they are 

 dipped in 10% formalin, drained, and 

 allowed to dry. They can then be 

 stored for long periods until required. 



Good sections 10 ju thick are easy to 

 cut by this method, and with practice 



