102 MICROSCOPIC TECHNIQUES 



2. Preparation of Tissues 



The preparation of sections from frozen dried material has the 

 advantage that soluble or diffusible constituents will have no chance 

 of being lost or displaced from their original sites. However, while 

 this is the method of choice, paraffin sections of formalin-fixed 

 tissue have been employed with success in certain instances, although 

 neither celloidin nor gelatin sections can be used since these media 

 give rise to fluorescence. 



When paraffin sections are employed, fixation is usually carried 

 out in 5-10% formalin for not longer than 24 hr. The sections are 

 cut 7-8 /x thick; egg albumin has been used to make them adhere to 

 the slides. The paraffin is removed by immersion in xylol for 30 

 min. and the sections are dried at room temperature. In this form 

 they have been kept for months, and may be examined without 

 further treatment. All reagents and materials should be the purest 

 obtainable to avoid adventitious fluorescence of contaminants. 



3. Photomicrography 



Photomicrographs of fluorescing preparations can be made if 

 certain precautions are taken. Popper and Elsasser (1941) found 

 that, in the photomicrography of vitamin A fluorescence, it is pref- 

 erable to use film of maximal daylight sensitivity. The Fluorapid 

 film of Agfa-Ansco Corp. is particularly well suited for the purpose 

 (Fig. 4C,D) . Kodachrome film can be used when the tissue is rich in 

 vitamin A, but the lower sensitivity of this film limits its value. In 

 general black-white film is preferable to the color variety. 



With substances of fading fluorescence, such as vitamin A, the 

 time employed for focusing must be kept to a minimum even at 

 the expense of the sharpness of the picture. The exposure itself must 

 be short (a maximum of 30 sec, regardless of magnification, in the 

 case of vitamin A) in order to obtain greater contrast between the 

 fluorescence and the background. Only one exposure can be made 

 as shown in Figure 4A,B. 



When the fluorescence does not fade, Kodachrome film can give 

 excellent results. The exposure time has to be determined by trial 

 in each case, usually falling in the range of 1-15 min. with an 

 average of about 2 min., according to Metcalf and Patton (1944). 

 The loss in intensity with greater magnifications makes it impracti- 



