LIPASE 



138 



LIPIODOL 



acetone, 12-24 hrs. each, room tempera- 

 ture. 



3. Impregnate in 5% acetylcellulose 

 (Eastman's cellulose acetate "high 

 acetyl, low viscosity, no. 4644") for 

 24 hrs. 



4. Drain off fluid, transfer to 2 

 changes benzene, 1 hr. each. 



5. Embed in paraffin (56-62°C), 2 

 changes, 1 to 1| hrs. each. Cut 4-8^ 

 sections, float on water (± 3o°C) and 

 mount on slides. Pass clown through 

 xylol and alcohols to aq. dest. 



6. Incubate at 37°C 6-12 hrs. in 50 cc. 

 Solution I + 2 cc. Solution II. 



Solution I: Glycerin 150 cc, 10% aq. 

 calcium chloride, 50 cc; half-molar 

 maleate buffer pH 7 to 7.4 (maleic acid, 

 5.8 gm. ; 4% aq. sodium hydroxide 94 cc. 

 -f aq. dest. 6 cc). If maleate buffer 

 is omitted mi.xture should be adjusted 

 to pH indicated. 



Solution II: 5% aq. Tween 40, or 

 Tween 60 (Atlas Powder Co., Wilming- 

 ton, Del.) or Product 81 with about 

 0.02% merthiolate added. Keep both 

 stock solutions in ice box. 



7. Rinse in aq. dest. and transfer to 

 1-2% aq. lead nitrate, 10-15 min. 



8. Rinse thoroughly in repeated 

 changes aq. dest. and transfer to dilute 

 solution of light yellow ammonium sul- 

 fide (few drops to Coplin jar of aq. dest.) 

 whereupon sites of lipase activity be- 

 come dark brown. 



9. Wash under tap and counterstain 

 with hematoxylin and very lightly with 

 eosin. 



10. Dehydrate in alcohols; clear in 

 gasoline or tetrachloroethylene (per- 

 chloroethylene) and mount in clarite in 

 these solvents. Avoid toluol and xylol. 



Lipids. Identification of various kinds in 

 microscopic preparations is extremely 

 difficult. As Lison (p. 192) has shown, 

 reliance cannot be placed in solubility 

 tests. Some bodies, soluble in alcohol, 

 ether, chloroform, carbon tetrachloride 

 and so on, are not fats while some fats 

 show considerable resistance to such sol- 

 vents. Formalin fixation itself causes 

 marked changes in solubility of fatty 

 bodies (Ivaufmann, C. and Lehmann, 

 E., Virchow's Archiv. f. Path. Anat. 

 und Physiol., 1926, 261, 623-648). It is 

 not unusual to find fats slightly soluble 

 or insoluble in microscopic preparations 

 which on chemical extraction are soluble. 

 Results of examination in polarized light 

 must, he states, be interpreted with 

 caution. Glj^cerides and fatty acids 

 examined in vivo are never birefringent 

 in the dissolved condition. After freez- 

 ing or treatment with formalin they can 

 become crystalline and birefringent. 

 Cholesterol, in the form of birefringent, 



rhombic plates, is of rare occurrence 

 in vivo, but easily recognizable. Choles- 

 terides appear sometimes as droplets 

 presenting the black cross of polarization 

 when viewed at low temperature. 

 When temperature is increased they lose 

 birefringence and look like droplets of 

 fat. Birefringence is lost as a result 

 of osmi cation. Coloration with sudan 

 and mounting in syrup of levulose 

 decreases birefringence. Lison gives 

 following tabular method of analysis 

 (abbreviated). 



1. In frozen sections, mounted in levulose syrup, 

 without artificial coloration, generally yellow 

 orange or brown. 



2. Iodine - iodide solution (like Gram's or 

 Lugol's) gives black -green or brown. Chromic 

 acid solution decolorizes quickly or slowly — 

 carotinoids. 

 2. Above reactions negative. Sulphuric acid 

 sometimes gives red color — chromolipoids. 

 1. In frozen sections show no natural color. 



2 Liebermann reaction (Schultze or Romieu 

 technique) positive: color blue, purple or violet, 

 becoming green. 



3. Digitonine reaction (Brunswick or Leulier- 

 Noel technique) gives crystals strongly illu- 

 minated between crossed nicols, unstainable 

 by histological methods — free cholesterol. 

 3. Digitonine reaction gives no crystalline 

 ppt. — cholesterides . 

 2. Liebermann reaction negative after repeated 

 attempts, no coloration or brown or red color. 

 3 Mounted in levulose syrup, without arti- 

 ficial coloration, examined with crossed 

 nicols, brightly illuminated and showing 

 cross of polarization— LipiTies. 

 3. Mounted in same way, without artificial 

 coloration, examined with crossed nicols, 

 not illuminated or illuminated but without 

 showing cross of polarization. 

 4. Smith-Dietrich reaction at 50°C. posi- 

 tive, color black— Lipines. 

 4. Above reaction negative. Coloration 

 gray or absent. 



5. Lorrain Smith reaction with nile blue 

 sulphate: rose — non-saturated glyc- 

 eride. 

 5. Above reaction absent or blue — Sat- 

 urated or non-saturated glyceride, or 

 fatty acid or Lipine. 



The much used Osmic Acid and Sudan 



staining methods are helpful when 

 other evidence is available as to 

 chemical constitution of substances 

 demonstrated. Fluorochromes are use- 

 ful for fluorescent visualization of fats 

 (Metcalf, R. L. and Patton, R. L., Stain 

 Techn.. 1944, 19, 11-27). See Fatty 

 Acids, Soaps, Neutral Fats (Glycerides), 

 Lipoids, Ciioiesterol (free). Cholesterol 

 Esters, Myeloidia, Myelin, etc 



Lipines, see Lipoids. 



Lipiodol, reactions in tissue to fat stains 



