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The Demonstration of Cutaneous Nerves*
Vol. 53, No. 5
THE JOURNAL 01' INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1969 by The Williams & Wilkins Co.
THE DEMONSTRATION OF CUTANEOUS NERVES* NICKOLAS A. ROMAN, A.Sc., DANIEL FORD, B.A. AND WILLIAM MONTAGNA, Ph.D.
When successful, the technique of Koelle and coarse acicular crystals form in it, making and Friedenwald (1) for cholinesterases, as the preparations fuzzy. We have now standmodified by Gomori (2), demonstrates both ardized the procedures so that preparations the localization of the enzymes and the pattern show predictably discrete localization and, with
', H
•
—
e
_t-
Fie. 1. Nerves around a vellus hair follicle from the eyebrow of man, prepared with the technique for AChe alone. The bracket indicates the extent of the follicle end-organ which shows a diffusion of enzyme activity. Ca. X 224.
of nerve distribution. However, the reaction
the superimposition of silver impregnation
product (cupric sulfide) often has a weak color
upon the cholinesterase techniques, the nerves appear a clean dark brown or black instead of
Received June 12, 1969; accepted for publication
June 16, 1969.
yellowish brown.
Primate Research Center, supported in part by
long and tedious, but the results are rewarding.
Publication No. 411 from the Oregon Regional
Grant FR 00163 of the National Institute of
The procedure described here may seem
Health and by funds from Grant AM 08445. Also
supported in part by funds from the Revlon Research Center, Inc., New York.
METHOI9
1. Use fresh tissues or unfixed tissues stored in * From the Department of Cutaneous Biology, Oregon Regional Primate Research Center, 505 dry ice or liquid nitrogen. Tissues may be kept N. W. 185th Avenue, Beaverton, Oregon 97005. safely in liquid nitrogen for several months with328
TECHNIQUE FOR NERVES
329
out an appreciable diminution in enzyme reactivity.
penclix) for acetylthiocholinesterase and butyryl-
This slight heating will do no harm.
are used.
few sections under the microscope and adjust the
6. Wash sections through 10 separate changes of saturated sodium sulfate. The length of time
tissue holder of the microtome accordingly.
for each washing is not critical.
thiocholinesterase for 2—5 hours at 37° C. Control
2. Cut frozen sections of unfixed tissue in a clyo-
reaction by adding Eserine (physostigmine salistat at thicknesses of 50, 60, 70, 80, and 100 and cylate) in concentration 10 to incubating mixstore them dry in a precooled screw-cap jar inside ture. the cryostat. Sections can also be affixed directly .fjote• Discrete localization without diffusion onto slides coated with Tissue-tac. They spread and avoidance of large crystals can be obtained evenly when a finger is placed on the underside. only if all reagents are prepared just before they
Note: To obtain good orientation, examine a
3. Pour cold 10% formaljn fixative into the jars containing sections or slides and place them for 2 hours in the refrigerator at 4° C. 4. Wash sections in 6 changes of distilled water at room temperature.
7. Dip each section or slide for 3 minutes in
freshly prepared 7% yellow ammonium sulfide.
8. Wash in 3 changes of distilled water, 2—3
minutes each.
9. Leave the sections in 10% formalin in the Note: Always handle free sections one at a refrigerator (4°C) overnight; they may be left time, with a glass rod bent and flattened like a in tormalin for loiger periods without harm. hockey stick.
Wash in 5 or more uhanges o distilled water before the next step.
Z.. fr?
t .L
5. Incubate in the buffered substratc (see Ap-
Fro. 2. From the same tissue as Figure 1, nerves around a vellus follicle prepared with the AChe-silver impregnation method described here. The bracket indicates the follicle end-organ. Compare with Figure 1. This preparation is 80 thick, and at any one focal plane many more nerves can be discerned than when the technique is not followed by silver impregnation. There are also many more nerves than shown in this photograph. Ca. X 260.
330
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
10. Defat sections and dehydrate through maximum of 10 seconds, the washing time may he
graded alcohols, starting with 30% alcohol to absolute, at least 20—30 minutes in each change. Trans-
increased or decreased. 16. Tone in 0.2% gold chloride for 3 minutes. 17. Rinse in 4 changes of distilled water.
fer to 2 changes of fresh xylol; one hour each. Rchydrate gradually to distilled water and place 18. Transfer to 5% sodium thiosulphate for 5 in Winkclmann's fixative (3, 4) overnight in the minutes. 19. Wash in 3 changes of distilled water.
refrigerator.
11. Wash sections in 6—8 changes of distilled
20. Dehydrate in a graded series of alcohols, clear, and mount in Permount.
water for a total of one hour or longer.
12. Dip the sections in 20% silver nitrate in a Note: Light countcrstaining is optional. Paracarpctri dish for 20 minutes. As with all silver im- mine in 70% alcohol during the final dehydration pregnation procedures, dishes should be scrupu- gives satisfactory results. lously clean. Each section must lie flat and free from folds. 13. Wash rapidly in 3 changes of distilled water,
COMMENTS
The advantage of this method is that it increases contrast and eliminates large crystals 14. Immerse each section gently in the hydro- in the reaction product. Even the finest single quinone and sodium sulfite solution for 10 min- nerve fibers arc demonstrated clearly as brown utes. Since sections tend to float to the surface, to black lines. While the preparations have the total washing time not to exceed 10 seconds. Handle every section separately.
coax them gently to lie flat on the bottom. 15. Rinse in 4 changes of distilled water (2—3 approximately the quality of silver impregna-
minutes in each). The washing time after silver tion techniques, it is likely that all nerves are nitrate impregnation is dependent upon the tis- in evidence around parenchymal tissues, whether sue and thickness of the section. If the sections or not they are demonstrated with silver
are too dark or light after the recommended
impregnation techniques alone. A comparison of Figures 1, 2, and 3 illustrates these points. The large concentrations of cholinesterases in cutaneous sensory end-organs still diffuse away from the original site, and there is a loss of fine details, as seen in Figures 1 and 2. This can be partially avoided by cutting short the incubation time in the substrate mixture. The manipulations described here are particularly valua-
ble for studying cutaneous nerves, many of which are difficult to show with silver impreg-
nation alone. We recommend that both thin and thick sections be prepared, so that the path of nerves can be followed accurately. APPENDIX
I
Preparation of Acetyl and But yrylt hiocholine Iodide Substrates
A. Stock solution 1. To 170 ml of distilled water, add 50 grams anhydrous sodium sulfate (Na2504), stir-
ring constantly. Warm the flask gently
Fic. 3. Nerves around a vellus follicle from the ear of man, prepared with the silver impregnation technique of Winkelmann alone. Only the nerves that emerge from the end-organ (in brackets) and the end-organ itself are outlined; the other nerves arc not argyrophilic. Ca X 223.
under hot tap water. 2. To the above solution add the following reagents in the order given, allowing each to dissolve completely before adding the next: Cupric sulfate (CuSOe 5110) 0.3 g 0.375 g Glycine (H2NCHrCOOH) Magnesium chloride (MgCl2 61120)
1.0 g
331
TECHNIQUE FOR NERVES
3. Adjust the stock solution to pH 5. 2 with C. 0.2 g hydroquinone and 1 g sodium sulfite in 100 ml distilled water. If the hydroquinone a maleic buffer (1.75 g maleic anhydride solution becomes cloudy during the stain[C4H203] in 30 ml 1 N Sodium hydroxide). ing procedure, change to a fresh batch. Store stock solution in 37 C oven until Have an excess of hydroquinone solution ready to use. Care in mixing and storing (covered) on hand, in case it is needed. this solution will eliminate crystal forD. 0.2% aqueous gold chloride. mation in the tissues. 13. Incubating medium (prepare just before E. 5% aqueous sodium thiosulfate. Except for the gold chloride, all solutions should be use) made fresh each time and should not be 1. Acetyl cholinesterase reaction: 20.0 mg acetyithiocholine iodide in 10.0
allowed to stand for more than several
ml of the stock solution heated to 37 C.
hours.
2. Butyryl cholinesterase reaction:
REFERENCES
25.0 mg butyrylthiocholine iodide in 1. Koelle, G. B. and Friedenwald, J. S.: A histo10.0 ml of the stock solution heated to chemical method for localizing cholinesterase activity. Proc. Soc. Exp. Biol. Med., 70: 617, 37 C. 1949. C. 7% Ammonium Sulfide (NH4)25) (prepare 2. Gomori, G.: Microscopic Histochemistry: Prinjust before use)
Preparation of Winlcelmann Reagents
A. Fixative—1.0 g ammonimn hydroxide
ciples and Practice. The University of Chicago Press, Chicago, Illinois, 1952.
3. Winkelmann, R. K.: A silver impregnation method for peripheral nerve endings, J. Invest. Derm., 24: 57, 1955.
(NH4OH) and 15 g sucrose in 100 ml 10% 4. Winkelmann, R. K.: Nerve EndinG's in Normal and Pathologic Skin: Contributions to the formalin. Make an excess of this solution. Anatomy of Sensation. Charles C. Thomas, B. 20% aqueous silver nitrate (AgNO3). Springfield, Illinois, 1960.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
THE JOURNAL 01' INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1969 by The Williams & Wilkins Co.
THE DEMONSTRATION OF CUTANEOUS NERVES* NICKOLAS A. ROMAN, A.Sc., DANIEL FORD, B.A. AND WILLIAM MONTAGNA, Ph.D.
When successful, the technique of Koelle and coarse acicular crystals form in it, making and Friedenwald (1) for cholinesterases, as the preparations fuzzy. We have now standmodified by Gomori (2), demonstrates both ardized the procedures so that preparations the localization of the enzymes and the pattern show predictably discrete localization and, with
', H
•
—
e
_t-
Fie. 1. Nerves around a vellus hair follicle from the eyebrow of man, prepared with the technique for AChe alone. The bracket indicates the extent of the follicle end-organ which shows a diffusion of enzyme activity. Ca. X 224.
of nerve distribution. However, the reaction
the superimposition of silver impregnation
product (cupric sulfide) often has a weak color
upon the cholinesterase techniques, the nerves appear a clean dark brown or black instead of
Received June 12, 1969; accepted for publication
June 16, 1969.
yellowish brown.
Primate Research Center, supported in part by
long and tedious, but the results are rewarding.
Publication No. 411 from the Oregon Regional
Grant FR 00163 of the National Institute of
The procedure described here may seem
Health and by funds from Grant AM 08445. Also
supported in part by funds from the Revlon Research Center, Inc., New York.
METHOI9
1. Use fresh tissues or unfixed tissues stored in * From the Department of Cutaneous Biology, Oregon Regional Primate Research Center, 505 dry ice or liquid nitrogen. Tissues may be kept N. W. 185th Avenue, Beaverton, Oregon 97005. safely in liquid nitrogen for several months with328
TECHNIQUE FOR NERVES
329
out an appreciable diminution in enzyme reactivity.
penclix) for acetylthiocholinesterase and butyryl-
This slight heating will do no harm.
are used.
few sections under the microscope and adjust the
6. Wash sections through 10 separate changes of saturated sodium sulfate. The length of time
tissue holder of the microtome accordingly.
for each washing is not critical.
thiocholinesterase for 2—5 hours at 37° C. Control
2. Cut frozen sections of unfixed tissue in a clyo-
reaction by adding Eserine (physostigmine salistat at thicknesses of 50, 60, 70, 80, and 100 and cylate) in concentration 10 to incubating mixstore them dry in a precooled screw-cap jar inside ture. the cryostat. Sections can also be affixed directly .fjote• Discrete localization without diffusion onto slides coated with Tissue-tac. They spread and avoidance of large crystals can be obtained evenly when a finger is placed on the underside. only if all reagents are prepared just before they
Note: To obtain good orientation, examine a
3. Pour cold 10% formaljn fixative into the jars containing sections or slides and place them for 2 hours in the refrigerator at 4° C. 4. Wash sections in 6 changes of distilled water at room temperature.
7. Dip each section or slide for 3 minutes in
freshly prepared 7% yellow ammonium sulfide.
8. Wash in 3 changes of distilled water, 2—3
minutes each.
9. Leave the sections in 10% formalin in the Note: Always handle free sections one at a refrigerator (4°C) overnight; they may be left time, with a glass rod bent and flattened like a in tormalin for loiger periods without harm. hockey stick.
Wash in 5 or more uhanges o distilled water before the next step.
Z.. fr?
t .L
5. Incubate in the buffered substratc (see Ap-
Fro. 2. From the same tissue as Figure 1, nerves around a vellus follicle prepared with the AChe-silver impregnation method described here. The bracket indicates the follicle end-organ. Compare with Figure 1. This preparation is 80 thick, and at any one focal plane many more nerves can be discerned than when the technique is not followed by silver impregnation. There are also many more nerves than shown in this photograph. Ca. X 260.
330
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
10. Defat sections and dehydrate through maximum of 10 seconds, the washing time may he
graded alcohols, starting with 30% alcohol to absolute, at least 20—30 minutes in each change. Trans-
increased or decreased. 16. Tone in 0.2% gold chloride for 3 minutes. 17. Rinse in 4 changes of distilled water.
fer to 2 changes of fresh xylol; one hour each. Rchydrate gradually to distilled water and place 18. Transfer to 5% sodium thiosulphate for 5 in Winkclmann's fixative (3, 4) overnight in the minutes. 19. Wash in 3 changes of distilled water.
refrigerator.
11. Wash sections in 6—8 changes of distilled
20. Dehydrate in a graded series of alcohols, clear, and mount in Permount.
water for a total of one hour or longer.
12. Dip the sections in 20% silver nitrate in a Note: Light countcrstaining is optional. Paracarpctri dish for 20 minutes. As with all silver im- mine in 70% alcohol during the final dehydration pregnation procedures, dishes should be scrupu- gives satisfactory results. lously clean. Each section must lie flat and free from folds. 13. Wash rapidly in 3 changes of distilled water,
COMMENTS
The advantage of this method is that it increases contrast and eliminates large crystals 14. Immerse each section gently in the hydro- in the reaction product. Even the finest single quinone and sodium sulfite solution for 10 min- nerve fibers arc demonstrated clearly as brown utes. Since sections tend to float to the surface, to black lines. While the preparations have the total washing time not to exceed 10 seconds. Handle every section separately.
coax them gently to lie flat on the bottom. 15. Rinse in 4 changes of distilled water (2—3 approximately the quality of silver impregna-
minutes in each). The washing time after silver tion techniques, it is likely that all nerves are nitrate impregnation is dependent upon the tis- in evidence around parenchymal tissues, whether sue and thickness of the section. If the sections or not they are demonstrated with silver
are too dark or light after the recommended
impregnation techniques alone. A comparison of Figures 1, 2, and 3 illustrates these points. The large concentrations of cholinesterases in cutaneous sensory end-organs still diffuse away from the original site, and there is a loss of fine details, as seen in Figures 1 and 2. This can be partially avoided by cutting short the incubation time in the substrate mixture. The manipulations described here are particularly valua-
ble for studying cutaneous nerves, many of which are difficult to show with silver impreg-
nation alone. We recommend that both thin and thick sections be prepared, so that the path of nerves can be followed accurately. APPENDIX
I
Preparation of Acetyl and But yrylt hiocholine Iodide Substrates
A. Stock solution 1. To 170 ml of distilled water, add 50 grams anhydrous sodium sulfate (Na2504), stir-
ring constantly. Warm the flask gently
Fic. 3. Nerves around a vellus follicle from the ear of man, prepared with the silver impregnation technique of Winkelmann alone. Only the nerves that emerge from the end-organ (in brackets) and the end-organ itself are outlined; the other nerves arc not argyrophilic. Ca X 223.
under hot tap water. 2. To the above solution add the following reagents in the order given, allowing each to dissolve completely before adding the next: Cupric sulfate (CuSOe 5110) 0.3 g 0.375 g Glycine (H2NCHrCOOH) Magnesium chloride (MgCl2 61120)
1.0 g
331
TECHNIQUE FOR NERVES
3. Adjust the stock solution to pH 5. 2 with C. 0.2 g hydroquinone and 1 g sodium sulfite in 100 ml distilled water. If the hydroquinone a maleic buffer (1.75 g maleic anhydride solution becomes cloudy during the stain[C4H203] in 30 ml 1 N Sodium hydroxide). ing procedure, change to a fresh batch. Store stock solution in 37 C oven until Have an excess of hydroquinone solution ready to use. Care in mixing and storing (covered) on hand, in case it is needed. this solution will eliminate crystal forD. 0.2% aqueous gold chloride. mation in the tissues. 13. Incubating medium (prepare just before E. 5% aqueous sodium thiosulfate. Except for the gold chloride, all solutions should be use) made fresh each time and should not be 1. Acetyl cholinesterase reaction: 20.0 mg acetyithiocholine iodide in 10.0
allowed to stand for more than several
ml of the stock solution heated to 37 C.
hours.
2. Butyryl cholinesterase reaction:
REFERENCES
25.0 mg butyrylthiocholine iodide in 1. Koelle, G. B. and Friedenwald, J. S.: A histo10.0 ml of the stock solution heated to chemical method for localizing cholinesterase activity. Proc. Soc. Exp. Biol. Med., 70: 617, 37 C. 1949. C. 7% Ammonium Sulfide (NH4)25) (prepare 2. Gomori, G.: Microscopic Histochemistry: Prinjust before use)
Preparation of Winlcelmann Reagents
A. Fixative—1.0 g ammonimn hydroxide
ciples and Practice. The University of Chicago Press, Chicago, Illinois, 1952.
3. Winkelmann, R. K.: A silver impregnation method for peripheral nerve endings, J. Invest. Derm., 24: 57, 1955.
(NH4OH) and 15 g sucrose in 100 ml 10% 4. Winkelmann, R. K.: Nerve EndinG's in Normal and Pathologic Skin: Contributions to the formalin. Make an excess of this solution. Anatomy of Sensation. Charles C. Thomas, B. 20% aqueous silver nitrate (AgNO3). Springfield, Illinois, 1960.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.