AXTIGENS, HISTOCHEMICAL 



17 



ANTIGENS, HISTOCHEMICAL 



phate at pH 7.0 in the cold until the 

 dialysate outside the sac shows fluores- 

 cence of less than 1 part in 20 million. 

 This can be roughly determined by eye 

 using a known solution of fluorescein as 

 a standard. 



The antibody solution may be further 

 purified by one or more precipitations 

 with half saturated ammonium sulphate 

 followed by re-solution and dialj^sis, 

 and by precipitation with cold acetone 

 (Coons, et al., J. Immunol., 1942, 45, 

 159) or by 40% alcohol (Marshall, J. 

 Exp. Med., 1951, 94, 21). Merthiolate 

 (Eli Lilly Co.) should be added as a 

 preservative (1:100 of a 1% solution). 



Despite these chemical purification 

 procedures, substances remain in such 

 fluorescein-protein conjugates which 

 stain some tissue elements. It has been 

 found necessary to shake such con- 

 jugates with dry acetone-powder de- 

 rived from animal tissues, usually liver 

 powder of the species whose tissue it 

 is proposed to study. Such powder 

 may be prepared by homogenizing the 

 tissue in a Waring blendor, washing 

 several times with distilled water in 

 the centrifuge, suspending the product 

 in saline and precipitating it with four 

 volumes of acetone. The acetone pre- 

 cipitate can be harvested on a Buchner 

 funnel and washed with dry acetone to 

 remove water. Such a powder is added 

 to a small aliquot of conjugate in the 

 proportion of 100 mgm. of powder to 

 each ml. of conjugate, the paste allowed 

 to stand for an hour at room temper- 

 ature, and the powder separated in the 

 centrifuge. The yield is higher if the 

 centrifugation is carried out in the cold 

 at 18,000 rpm. Two such absorptions 

 are often necessary to remove "non- 

 specific staining." Merthiolate should 

 be added again as above. 



The preparation of tissue sections for 

 use ivith fluorescent antibody. The prob- 

 lem of preparing tissue sections retain- 

 ing the antigenic activity of the ma- 

 terial sought varies with the antigen in 

 question. The bacterial polysaccha- 

 rides which have so far been studied 

 survived fixation in Rossman's picric 

 acid-alcohol-formalin followed by 

 paraffin embedding. Such sections are 

 deparafRnized and hydrated and then 

 stained with the appropriate antibody 

 solution. Care must be taken not to 

 wash out the antigen during the pro- 

 cedures preceding the flooding of the 

 section with labeled antibody. In the 

 case of bacterial polysaccharides, it is 

 necessary to remove the picric acid in 

 70% alcohol, in which these polysac- 

 charides are insoluble. 



In the case of less stable materials, 



for example proteins, fixation of the 

 tissue-block is unsatisfactory; sections 

 must be prepared from unfixed material 

 and fixation carried out on the indi- 

 vidual section. There are two methods 

 available for the preparation of such 

 sections from unfixed tissue, one that 

 of Linderstr0m-Lang and Mogensen, 

 the other that of Altmann-Gersh, and 

 others. 



With either of these two methods, 

 it is necessary to fix the tissue section 

 in some appropriate reagent before 

 applying labeled antibody solutions 

 lest the material looked for be dissolved 

 out of the section during exposure to 

 conjugated antibody. A certain 

 amount of experimentation is necessary 

 with each new antigen in order to find 

 the appropriate fixative. For mumps 

 and influenza A virus and the virus of 

 infectious canine hepatitis, acetone is 

 satisfactory. This is used at room 

 temperature for 15 to 30 minutes fol- 

 lowed by drying of the section in an 

 incubator. For proteins, 95% ethanol 

 by volume (start with absolute ethanol) 

 at 37°C. for 30 minutes is satisfactory. 

 For ACTH in the anterior lobe of the 

 hog pituitary, absolute methanol has 

 been shown to be a satisfactory fixative 

 (Marshall, J. Exp. Med., 1951, 94, 21). 

 In general, these organic solvents are 

 best removed by evaporation. 



These fixatives and exposure-times 

 are cited as examples only. Any anti- 

 gen-antibody system chosen for study 

 must be investigated from this as well 

 as from other points of view. 



The use of labeled antibody solutions 

 on tissue sections. A small drop of 

 fluorescein-antibody solution is placed 

 over the tissue section, and allowed to 

 react for from 10 minutes to 48 hours, 

 depending on the system under study. 

 Thirty minutes is usually satisfactory. 

 During the reaction, evaporation must 

 be minimized. Usually the reaction 

 can be carried out at room temperature, 

 although incubator temperatures or 

 refrigerator temperatures can be em- 

 ployed. Following exposure to the 

 fluorescent antibody solution, the sec- 

 tion should be placed in a Coplin jar 

 containing 0.9% saline buffered at pH 

 7.0 with 0.01 molar phosphate, and 

 washed with very gentle motion for 

 10 minutes. At the end of this time, 

 the slide should be wiped dry except 

 for the area of the section and the sec- 

 tion itself mounted under a cover slip 

 in glycerol containing a trace of buffer 

 at pH 7 (commercial glycerol is slightly 

 acid), and examined under the fluores- 

 cence microscope. 



Control of specificity of staining. 



