PERMEABILITY 



190 



PEROXIDASE 



Covell (Anat. Rec, 1928, 40, 213-223) 

 which show secretory products leaving 

 the cell in protrusions of the plasma 

 membrane. These later become 

 pinched off, the membranes disintegrate 

 and the product is set free in the lumen. 

 See literature review (Blinks, L. R., 

 Ann. Rev. Physiol., 1942, 4, 1-24). See 

 Spreading Factors. 

 Peroxidase. This enzyme catalyses oxida- 

 tion of several oxidizable substrates in 

 presence of peroxide. It is most abun- 

 dant in plants being usually prepared 

 from horse-radish. In mammals it 

 occurs in mammary glands and in milk. 

 In the -peroxidase reaction, so commonly 

 employed in the study of leucocytes, a 

 colored product is formed in the pres- 

 ence of peroxide from a suitable sub- 

 strate, benzidine or alpha naphthol. 

 Blaschko and Jacobson (Bourne, p. 197) 

 remind us that it is still uncertain that 

 this reaction in leucocytes demonstrates 

 a true peroxidase because it is relatively 

 stable to heat. 



1. Alpha naphthol-pyronin (Gra- 

 ham, G. S., J. Med. Res., 1916, 30, 231- 

 242). Fix blood smears in 9 parts 95% 

 alcohol and 1 part formalin freshly pre- 

 pared, 1-2 min. Wash in water and 

 flood with : alpha naphthol (Merck's 

 "recrystallized" or "Reagent"), 1 gm.; 

 40% alcohol, 100 cc. ; hydrogen peroxide, 

 0.2 cc. for 4-5 min. Wash in dish of 

 running water, 15 min. Stain in: 

 pyronin 0.1 gm.; anilin oil, 4 cc; 40% 

 alcohol 96 cc, 2 min. Wash in water. 

 Stain in 0.5% aq. methylene blue 

 (Griibler's BX), \-l min. Wash in 

 water, blot, mount in neutral balsam. 

 Fresh smears should be used. When 

 used by a class of students tie droppers 

 to bottles to avoid spoiling solutions by 

 mixing tliem. 



2. Benzidine-methylene blue (Gra- 

 ham, G. S., J. Med. Res., 1918, 39, 15- 

 24). Fix as above. Wash in water. 

 Treat 5-10 min. in 0.2% hydrogen 

 peroxide in 40% alcohol saturated before 

 using with benzidine, 5-10 min. Wash 

 and stain with methylene blue. 



3. Benzidine-safranin (Sato, A. and 

 Shoji, K., J. Lab. and Clin. Med., 1927- 

 28, 13, 1058-1060). Dry blood smear 

 in air. Flood the slides with solution 

 A (0.5% copper sulphate). After 1 

 minute pour off solution but do not 

 wash or dry slides. Apply solution B 

 (rub up in a mortar 0.2 gms. benzidine 

 with a few drops distilled water. Then 

 add 200 cc. aq. dest. and filter. To 

 filtrate add 4 drops 3% hydrogen 

 peroxide) for 2 min. Then wash in tap 

 water. Stain with solution C (1% 

 safranin in aq. dest), 1 min. Wash in 

 tap water and dry. Peroxidase granules 



are colored blue in granular leucocytes 

 and the nuclei orange red. 



4. Nitroprusside-benzidine (Goodpas- 

 ture, E. W., J. Lab. & Clin. Med., 

 1919, 4, 442-444). To make the stain 

 dissolve 0.05 gm. sodium nitroprusside 

 in 2 cc aq. dest. ; add 100 cc. 95% alco- 

 hol ; 0.05 cc. benzidine C.P.; 0.05 gm. 

 basic fuchsin and 0.5 cc. hydrogen 

 peroxide. Cover well dried blood smear 

 with known amount of stain, 1 min.; add 

 equal volume aq. dest. plus hydrogen 

 peroxide, 3-4 min.; rinse thoroughly in 

 water and blot dry. Shows many blue 

 granules in granular leucocytes and few 

 in monocytes. Nuclei are colored red. 

 To increase intensity of stain dilute 

 with a little less aq. dest. and stain 

 longer. Method can be used for frozen 

 sections of material fixed in formalin 

 and preserved in 80% ale A modifica- 

 tion of this stain has been proposed by 

 Beacom (J. Lab. & Clin. Med., 1925-26, 

 11, 1092-1093) with hydrogen pero.xide 

 omitted and basic fuchsin doubled. 



5. Benzidine-Giemsa (Armitage, F. 

 L., J. Path., 1939, 49, 579-580). Fix 

 smears in 96% alcohol containing 10% 

 formol freshly made up. Flood with 

 benzidine mixture (0.75 gm. benzidine 

 in 500 cc. 40% ethyl alcohol. Filter. 

 Add 7 cc. 3% H2O2, mix by shaking im- 

 mediately before using) 2 min. for fresh 

 films, longer for older ones. Wash in 

 40% alcohol until definite yellow gran- 

 ules are seen in granular leucocytes. 

 Absolute alcohol and dry in incubator. 

 Counterstain with dilute Giemsa, wash 

 in water, blot and dry. 



6. Benzidine for paraffin sections 

 (McJunkin, F. A., Anat. Rec, 1922-23, 

 24, 67-76). After fixation of small 

 pieces in 10% formalin imbed quickly 

 in paraflRn; 70% alcohol, 1 hr. ; acetone, 

 30 min.; benzol, 20 min.; paraffin, 20 

 min. Mount thin sections in usual 

 fashion. Deparaffinize in benzol 20 

 sec, acetone, 10 sec. Water, few sec- 

 onds. Drain off water, apply mixture 

 (80% alcohol, 25 cc ; benzidine, 0.1 gm. ; 

 hydrogen peroxide, 2 drops) diluted with 

 1 or 2 parts aq. dest., 5 min. Water, 5 

 min.; hematoxylin, 2 min.; water, 1 

 min., 0.1% aq. eosin, 20 sec; 95% 

 alcohol, 30 sec. ; abs. alcohol, 5 sec. Clear 

 in xylol and mount in balsam. 



Note : In above methods a blue 

 counterstain tends to obscure the blue 

 peroxidase reaction. 



7. DCPIP-2, 6-dichlor-phenol-mdo- 

 phenol (Jacoby, F., J. Physiol., 1944, 

 103, Proc Physiol. Soc July 29). Fix 

 air dried bloocl smear in 9 parts abs. ale 

 and 1 part formol for 2-3 min. Wash in 

 water. Treat smear for 3-5 min. with 

 0.5% aq. 2.6-dichlor-phenol-indophenol 

 to every 5 cc. of which 4 drops H2O2 



