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Histochemical identification of enzyme activity in frozen tissues and tumors
CRYOBIOLOGY Vol. 1, No. 2, 1964
HISTOCHEMICAL IDENTIFICATION OF ENZYME ACTIVITY IN FROZEN TISSUES AND TUMORS” t P. J. MELNICK University
of California Medical Center, San Francisco; and Veterans Administration Hospital, Martinez, California
For those who work with the microscope, histochemical techniques open a large and fascinating area of research, present exceptional opportunities for a deeper understanding of disease, and are beginning to offer a small but useful number of practical diagnostic aids. The First’ International Congress of Histochemistry and Cytochemistry was held in 1960, and the Second in 1964, marking the recognition of a young specialty whose active growth began scarcely 25 years ago.‘. ’ Its basic principles had, however, been announced more than a century ago by Raspail. The techniques of histochemistry consist of chemical reactions produced in tissue sections, whose visible end-products seen through the microscope, are morphological expressions of the chemical make-up of cells, and of the function of t,heir enzymes. The requirements of a histochemical technique are the following: The method should be specific for the substance or enzyme identified; an easily visible, preferably colored, material or precipitate should be formed so that it is clearly seen through the microscope; and the chemical reaction should be very rapid so that no diffusion occurs and good localization results. The recent rapid expansion of histochemistry justifies a word of caution and conservatism. It will probably be a long time before standard conditions will be generally agreed upon for most histochemical procedures because the chemical react’ions carried out in the tissues are oft’en subtle, and occur in close proximity to many variable and reactive structures in the cells. Controls of several kinds are necessary. * Presented at the First Annual Meeting, Society for Cryobiology, August 24-26, 1964, Washington, D. C. ‘r Aided by U. S. Public Health Service Research Grant CA-07468, and a Veterans Administration Research appropriation. The generous cooperation of the Linde Company is gratefullv acknowledged, as well as the technical assistance of Pauline Heizer. Ph.D.. and the late Carl W. Howell.
False negative reactions may occur when chemical substances are masked or are bound in nonreactive form, when enzymes are held in an inactive state, or when tissues are not properly prepared for the procedure. An example of the latter is loss of soluble enzymes into the incubating solution. False positive reactions are not uncommon because of nonspecific chemical reactions, to enzymes acting on intrinsic unknown substrates, to nonenzymatic reactions of indicators, or to nonoptimal freezing rates of the tissues. Quantitation in histochemistry is very difficult; a feebly active enzyme may appear to be intensely active if it is permitted a long incubation period and thereby a large amount of end-product accumulates in the cells. There is need to check histochemical methods with appropriate biochemical, biophysical, or immunological techniques. There is also need to make comparative studies of the human with a variety of animal species. Most histochemical techniques were developed on normal animal tissues, and the optimal conditions for human tissues may often be different’. This is especially significant for enzymes, because the action of enzymes depends so largely on their precise genet,ically determined structure. Additional variables contributed by disease processes will need to be evaluated in the future. Despite all these complexities, a well defined amount of histochemical knowledge has accumulat,ed that has so far remained intact. In t’he past, histological methods have demonstrated cells merely by coloring them, but this gave only very general notions of the chemical makeup of the structures that were stained, such as basophilia of nuclei or acidophilia of cytoplasm. Today’s histochemical methods are being developed with a view toward demonstrating more precisely the chemical nature of the various structures within cells and of cell products. Methods have been devised to demonstrate various enzymes, carbohydrates, proteins, pigments, nucleic acids, inorganic substances, and
140
:HISTOCHEMICdL
IDENTIFICBTION
lipids. Since histochemistry and cryobiology are both young specialties, we are presented with many problems that require our careful attention.
ENZYMES One of the most exciting developments is the morphological demonstration of enzyme activity within cells. Enzymes are not “living” molecules; they are chemically active protein molecules that depend for their specific activity on their precise structure which is complementary to the structure of the substrates on which they act. If they become denat,ured to such an extent that the structure of their active sites is seriously changed, one rnay still be left with protein molecules, but they are no longer enzymes. Therefore, if ti,ssues can be so processed that their active sites are intact, enzyme activity is often retained. When sections of such tissues are placed into a solution containing a substrate, the specific enzyme, if present, may carry out its chemical action on the substrate if the proper conditions are present, such as pH, temperature, activating ions, and absence of inhibitors. If there is present in the solut,ion a substance with which a product of the reaction will combine to form an insoluble reaction product that can be easily seen with the microscope, the basis of a histochemical t,echnique is present. Most of t,he hydrolases are fairly sturdy and can withstand procedures such as formalin fixation at 5°C. In contrast to the hydrolytic enzymes, most of the dehydrogenases are very unstable in t,issues and will not withstand these procedures, the-refore they are generally studied only in fresh-frozen sections. Freeze-substitution methods are also useful, and freeze-dry methods, although elaborate, may also become practical in the fut’ure. Development, of a histochemical technique for alkaline phosphatase by Gomori in 1939 (and independently by Takamatsu) furnished a strong impetus for the rapid growth of enzyme histochemistry, indeed, for all of histochemistry. Gomori’s technique was the by now well known metal-salt meth.od, and was rapidly followed by similar methods for ot’her phosphatases such as 5’-nucleotidase, glucose-6 acid phosphatase, phosphatase, phosphamidase, and adenosine triphosphatase, for which Gomori’s principle has furnished the basis. The field was greatly ex-
OF ENZYME
ACTIVITY
141
panded when extensive use began to be made of naphtholic substrates for the hydrolytic enzymes. When the enzyme splits off the specific substit’uent from a naphtholic substrate, free naphthol is present. If a diazonium salt has been added to the incubating solution it, couples with the naphthol to form an azo dye that can be easily seen with the microscope as a colored precipit’ate. Today, naphtholic substrates have b een synthesized to demonst’rate very many hydrolaees. Postcoupling and other methods have been devised for certain enzymes, but for most hydrolases t’he simultaneous capture methods with the use of naphtholic substrates are the most widely used. A similar great advance was made in the histochemistry of t’he dehydrogenases by the use of tetrazolium salts. Specific dehydrogenases transfer electrons from their specific substrates, i.e., lactic acid, glutamic acid, etc., to diphosphopyridine nucleotide (DPN) or triphosphopyridine nucleot,ide (TPN) in most cases. In living cells, diaphorases transfer the electrons from the reduced DPN or TPN to the cytochrome chain and thence to molecular oxygen; and the energy released thereby becomes available chiefly for oxidative phosphorylation, i.e., for synthesis of that versatile molecule adenosine triphosphate (-4TP) from adenosine diphosphate ($DP). In t’he histochemical procedure the electrons are, instead, diverted from the reduced DPX or TP?; to a colorless tetrazolium salt added to the solution, which becomes reduced to an insoluble, intensely colored precipitate of formazan. Sometimes respiratory inhibitors such as cyanide are added to t)he solution to inhibit the cytochromes, to ensure that only the tetrazolium is active as an electron acceptor. Sometimes adjuvants such as phenazine methosulfate are added to facilitate the diversion of the electron flow to the tetrazolium, from such enzymes as euccinic dehydrogenase. Tetrazolium salts for dehydrogenase histochemistry have been greatly improved in recent years with the synthesis of nitro-blue tetrazolium and related compounds. The oxidases such as cptochrome oxidase, peroxidase, tyrosinasc system, and monoamine oxidase include a small miscellaneous group whose techniques are gradually being perfected. Histochemistry may become an effective means of studying neoplasme, because t’he great
142
P. J. MELNICK
genetic variability of tumors is reflected in their variable enzyme patterns. It is conceivable that the time may come when surgeons, upon submitting biopsies of malignant tumors to pathologists, will request not only the histological diagnosis, but also a histochemical study of the enzyme patterns, t,o help select the indicated chemotherapy. Similarly, studies of the enzyme patterns of chronic granulomas and other inflammations may some day perhaps add to the effectiveness of antimicrobial therapy. PLAN
OF STUDY
Between 1958 and 1961 a group of about 25 breast cancers and 31 lung cancers were studied with histochemical enzyme methods for 12 hydrolases and 14 oxidative enzymes.‘.’ The results of these studies were reported in detail elsewhere.3’ 4 The outstanding findings consisted, essentially, of enzyme profiles in the differentiated cancers that tended to resemble those of their normal t,issue cells of origin. The undifferentiated cancers, however, presented man) enzyme defects; and t,he more undifferentiated the cell type the fewer enzymes they contained, much like embryonal tissue. Of special interest were the many enzyme delet,ions encountered among the dehydrogenases, since these are so important in the bioenergetic pathways for furnishing controlled chemical energy upon which all life depends. Dehydrogenases must be examined in fresh frozen sections of the tissues, since they do not withstand such procedures as formalin fixation, and even TABLE CODE FOR IDENTIFIC~~TION TO WHICH CODE:
1. 2. 3. 4. 5. 6.
MATERIALS
AXD
METHODS
In 1962, a comparison was begun of the effect of various freezing rates on t’he histochemical identification of enzyme activity. Through the generous cooperat,ion of Dr. Arthus P. Rinfret of the Linde Company, under whose direction the various tissues were prepared and frozen, and shipped in a liquid nitrogen refrigerator to the author’s laboratory, 48 specimens of normal guinea pig liver and kidney were examined. Table 1 contains the code that identifies the tissues and the freezing modalities. Tables 2 and 3 present a compilation of the findings extracted from the large amount’ of detail, for 9 hydrolases and 8 oxidative enzymes. For the purposes of this comparative study, it was decided to use cryost,at sect,ions for bot,h the hydrolases as well as the dehydrogenases. The frozen sections are excellent for both groups of enzymes. In addition, during the past t#hree years, 1
OF NORM.~L GUINEA PIG KIDNEY AND LIVER AND THE CRYOGENIC THEY WER.E EXPOSED (COURTESY OF THE LINDE COMPANY)
MODALITIES
Describes sample type and cooling regime, as follows:
refers to sample type Kidney, l-mm slices Kidney, 2-mm slices Kidney, chunks (3 to 5 mm) Kidney, whole Liver, 2-mm slices Liver, chunks and whole lobes
NUMBER:
a few hours at room temperature will often inactivate them. As was mentioned above, nonoptimal freezing rates present one of the problems that require att’ention in the further development of histochemistry. It seemed possible, therefore, that inactivation of enzymes, by nonoptimal freezing rates in t,he course of making cryostat sections for the study of the dehydrogenases, was t’he basis for at least some of the enzyme deletions found. Problems such as these are increasingly occupying the attent,ion of histochemists.‘, *
refers to cooling regime A. Immersion in liquid Ns(-196°C) B. Immersion in Dry Ice-ethanol (SD-l) at -78”C, followed by immersion in liquid N, C. Immersion in Dry Ice-ethanol (SD-l) at -78”C, storage for one week in dry ice at -78”C, followed by immersion in liquid Nz D. Immersion in Ucon 12 (CClzF,) at -3O”C, followed by immersion in liquid Nz E. Immersion in Ucon 12 at -30°C storage for one week at -25°C (freezer), followed by immersion in liquid Nz F. Cooling to 50°C at approximately 1°C per minute in controlled rate freezer (BF-3), followed by immersion in liquid NB
LETTER:
HISTOCHEMICAL
IDENTIFICATIOX
OF ENZYME
TABLE NORMAL
GLXNE.I
PIG
KIDNEY
143
ACTIVITY
2
AND
LIVER
(CODE
IN
TABLE
1)
Hydrolases
:sterase
Cholinesterase
;lucose-6 Pa%2
TPase
-Gluuronidase I-
Liquid nitrogen (-196°C) 1A 2A 3A 4A 5A 6A Dry Ice-ethanol (-78°C) followed by liquid nitrogen 1B 2B 3B 4B 5B 6B Dry Ice-ethanol (-78°C) 1c 2c 3c 4c 5c 6C “Ucon” (-30°C) followed by liquid nitrogen ID 2D 3D 4D 5D BD “Ucon” (-30°C’ /) IE 2E 3E 4E 5E 6E Slow freeze at -1°C per minute 1F 2F 3F 4F 5F 6F
: + :zk
+
: f+ f+ T + f + f T + f f+ f f f 0 f 0
0 0 0 0 0 0
+ +
: f
z
+ f
1
+
+ +
: 0
z
+ 0
+
1
:
+
+ z 1 0
+
:
:
+
+
+ +
: f
+ +
f *
z
f 0
z
0
:
f
0 0
z
0 0
:
0 0
many examples of a number of normal human tissues obtained at autopsy or from surgical specimens, were similarly examined. Tables 4 and 5 summarize the findings in this mat’erial. The details of the histological techniques used in these studies have been reported in two other communications that describe chiefly the effect’
:
of various freezing rates on histochemical demon&ration of enzymes in tumors.3. 4 Essentially the same principles and findings apply to the tumor material, and the preliminary study of the normal tissues therefore furnished a valuable orientation for the further work on the tumors.
144
P. J. MELNICK TABLE NORMAL GUINEA PIG KIDNEY
3
AND LIVER
(CODS IN TABLE 1) Dehydrogenases
ic
Liquid nitrogen (-196°C) 1A 2A 3A 4A 5A 6A Dry Ice-ethanol (-78°C) followed by liquid nitrogen 1B 2B 3B 4B 5B 6B Dry Ice-ethanol (-78°C) 1c 2c 3c 4c 5c 6C “Ucon” (-30°C) followed by liquid nitrogen ID 2D 3D 41) 5D 6D “Ucon” (-30°C) IE 2E 3E 4E 5E GE Slow freeze at -1°C per minute 1F 2F 3F 4F 5F 6F RESULTS
During the past three years, following the preliminary study on the normal animal and human tissues mentioned above, 140 human tumors were studied in the following manner. Each tumor was cut into several aliquot blocks averaging about 4 mm thick. Several blocks
3lutamic Acid
>
G
Cytochrome Oxidase
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + : 0 0 0 f 0 f f f : : z : + +
were used for other purposes including the effect of fixation, examinat.ion for lipids and for glycogen, etc., but, for the purpose of this cryogenic study, first, one block was frozen rapidly by quenching it in isopentane cooled to -160°C by liquid nitrogen; second, one block was frozen at the rate of -1°C per minute in a
FIGS. 1 to 4 skeletal muscle, rapidly frozen. Cytochrome oxidase, Burstone method. x300. Activity is feeble, and there are scattered focal aggregates of cytochrome c activit,y, probably as a result of crystallized intrinsic cytochrome c due to freezing. FIG. 2. Normal skelet,al muscle, rapidly frozen. Cytochrome oxidase, Burstone method, with added cytochrome c. X300. Activity is intense and uniform. FIG. 3. Normal liver, rapidly frozen. Cytochrome oxidase, Burstone method. X150. Actil-ity is feeble, and there are aggregates of cytochrome c activity, probably as a result of crystallized cytochrome c probably separated out as a result of freezing. FIG. 4. Normal liver, rapidly frozen. Cytochrome oxidase, Burstone method, with added cytochrome c. Sctivity is uniform throughout.
FIG. 1. Normal
145
FIGS. 5 to 8 FIG. 5. Sormal jejunum, rapidly frozen. Cytochrome oxidase, Burstone method. Activity is essentially nonexistent, and is represented by the focal aggregates described above. FIG. 6. Normal jejunum, rapidly frozen. Cytochrome oxidase, Burstone method, with added cytochrome c. Although the focal aggregates of activity persist in this tissue that was quenched at -160°C in isopentane, enzyme activity is seen also in the jejunal epithelium. FIG. 7. Metastasis of carcinoma of the pancreas to the liver, slowly frozen. Succinic dehydrogenase. ~175. Ice crystal artefact is evident. Although activity is seen in a number of the liver cell cords, no enzyme activity is evident in the tumor. FIG. 8. Metastasis of a carcinoma of the pancreas to the liver, rapidly frozen. Succinic dehydrogenase. X17.5. Enzyme activity is seen throughout. This is an example of diminished enzyme activity in slow frozen tissue.
146
HISTOCHEMICAL
IDENTIFICATION
OF ENZYME
ACTIVITY
FIGS. 9 and 10 FIG. 9. Metastasis of an undifferentiated carcinoma of the lung to the liver, slowly frozen. Glutamic dehydrogenase. X125. Although some activity is seen in the liver there is no
enzyme activity in the metastatic tumor. FIG. 10. Metastasis of an undifferentiated carcinoma of the lung to the liver, rapidly frozen. Glutamic dehydrogenase. X125. Although the enzyme activit,y in the liver is enhanced, there is still no activity in the metastatic tumor, an example of a true enzyme deletion. controlled rate Linde BF3 liquid nitrogen freezer; and third, a third block was frozen on a cryostat chuck, whose freezing rate measured with a therm&or varies from -7” to -12°C per minute. Cryostat sections were cut from each block and stored in a deep freeze at -30°C until processed, usually within less than one month. Sections from all three blocks were incubated under identical conditions in the same container, the same incubating solution, and the same length of time. Reference to the reports of this study”, 4 will indicate that when enzyme activity in a given tumor was intense (such as acid phosphat,ase in prostatic cancers), no appreciable difference could be det’ected in the histochemical enzyme product at the three different freezing rat,es. The above described finding should be kept in mind in the interpret,ation of the tables that contain the data on the effect of various freezing rates on normal animal tissues, which generall:,
contain intact, intensely active enzymes, and which would not be expected to have enzyme deletions. As can be seen from the tables, for most of the enzymes strong activity was seen at all freezing rates. However, two types of findings are seen that encouraged us to continue this comparative cryogenic study in tumors, which might be expect’ed to have occasional weakly active enzymes in which differences might be accentuated by different freezing rates, and which also might be expected to have enzyme delet,ions. These two interesting findings in t’he normal tissues are as follows: first, slow freezing tended to diminish or to eliminate activity of the esterases, cholinesternses, certain lipases, and acid and alkaline phosphatnses, whereas rapid freezing preserved their full range of activity int,act ; second, rapid freezing generally abolished, or almost completely abolished, cytochrome c oxidase activity, whereas slow freezing
TABLE 4 CRYOENZYMOLOGY OF NORMAL HUMAN TISSUES
-
Code*
Hydrolases
-
-
Cholin :sterasce 1Lipase
E
A
.-
S~ucleo-
‘?2
A TPase
tidase
2+ 2+
2-t 2+
4+ 4f
3+ 3+
4f 4+
4+
A B
4+ 4+
2+ 2+
3+ 3+
0
4+ 4+
A B
4f 4+
3t 3+
0 0
2+
Skeletal IIlUS&
A B
3-k 3+
2+ 2+
0 0
Brain
A B
2+
1+
4+
3+
A B
2+ 4-t
A B A B
Myocardium
Nerve Pancreas Jejunum
Sulfatase
.-
4+ 4f
Liver
cumoidase
-
eucim :P tlanim AmiAminax nase
--
A B
Kidney
/B-GlU
4+
4+ 4-k
3f 3+
2+ 2+
4+ 4+
4+ 4+
4+ 4+
4+ 4+
4+ 4+
3+ 3f
4+ 4+
1+ 1+
4-t 4+
4+ 4t
1+ 4+
3+ 3+
1+ 1+
4+ 4+
2+
1+ If
2+ 2+
4+ 4+
1+ 1+
2+ 2+
1+ 1+
4+ 4+
0 1+
2+ 2+
2+ 3+
4+ 4+
If
it
2+
1+ 1+
1+ If
4+ 4+
1+ 3+
0 1+
2+ 2+
0 1+
4f 4+
2+
t:
4+
1+ 1+
1+ 1+
2:
4+ 4f
3+ 3+
3f 3+
3+ 3+
3+ 3+
4f 4f
4+ 4+
3+ 3f
2+ 4+
2+ 2+
4+ 4+
4+ 4+
3+ 3+
4+
4+ 4+
3+ 3+
4+ 4f
4+ 4+
2+ 2+
1+ 4+
1+
2+
4+ 4+
* Code: A = slow fr’ eeze;
w 4+ If
I 4+ I
5 = rapid
freeze.
-
-
-
TABLE 5 CRYOENZYMOLOGY OF NORMAL HUMAN TISSUES Dehydrogenases
Code*
suc-
SUCchic
2+
-
30 74;
cink system
IScitric
Glutami
GluEth: I- coseml I 6P
I acti c
coni
“GE-$8
2” g.2 20 u
2% es” 26 k” u
A B
4+ 4+
2+
4+ 4f
4+ 4+
I: 2
2
3+
3t 3+
2+ 2+
0 0
4+ 4f
4+ 4t
3t 0
4+ 4f
A B
4+ 4+
3+ 3+
4f 4+
4f 4t
3+ 3+
3+ 3t
4+ 4+
4+ 4+
4f 4+
3+ 4+
1-t
w
4+ 4+
4+ 4+
2+ i
4+ 4+
A B
4+ 4f
2+
4+ 4+
4+ 4+
3+ 3+
3+ 3+
2+
2+
4+
3+
4+ 4+
1-t 3+
0 0
4+ 4+
4+ 4+
3+ f
4+ 4+
Skeletal muscle
A B
2-t
2+
2+ 4+
1-t 3+
2+ 2+
1+ 3+
1+ 3+
2+
4+ 4t
;I
0 0
4+ 4+
3+ 3+
2+
4+
0
4+ 4+
Brain
A B
1+ 4+
0
2+
3+ 3+
4t 3-t
2+
2+
2+
2+
3+
4+
4+
3+
1+ 3+
0 0
3+ 3+
2+ 2+
3+ f
4+ 4+
A B
1+ 4f
0
1+ 3+
3+ 3+
1-t 3f
1+ 3+
a4+
2+ 3t
1+ 3+
0 0
3+ 3+
1+
4+
2+
3+ i
4+ 4+
Pancreas
A B
4+ 4f
3+ 3+
4+ 4f
4f 4+
4+ 4-f
4+ 4+
4+ 4+
4+ 4+
4+ 4+
4+ 4+
0 0
4+ 4+
4+ 4t
3+ 1+
4f 4+
Jeiunum
A B
3f 3f
2+
2+
2+
2+
4+
4+
4+
4+ 4f
t=
0 0
4+ 4+
4+ 4-k
2+
3+
1+ 3+
2+
4+
0
4+ 4+
Kidney Liver Myocardium
Nerve
4+ 4+ 1- r * Code: A = SIOW Iret
a+ 1+ 2+ 2+
4+
2+
e; I i := ra
2+
1+
-
Id freeze. 148
HISTOCHEMICAL
IDENTIFICATION
rates tended to :retain at least partial activity, especially in the whole livers and kidneys. The reader is referred to the two other reports on other aspects of the cryoenzymology of the 140 tumors studied3# ’ for discussions and principles brought out by this material. Summarized briefly: first, addition of cytochrome c to the incubating solution brought out full activity of the enzyme in all tissues, because freezing apparently crystallizes and sequestrates intrinsic cytochrome c making it unavailable as a substrate; seconsd, in the case of weakly active enzymes, slow freezing rates fail to bring out activity, leading to possible wrong interpretations of enzyme deletions, whereas quenching in isopentane at -160°C brings out the full range of activity; third, by such an approach determinations of true enzyme deletions in tumors can perhaps be est’ablished; and fourth, histochemical enzyme techniques should be able significantly to supplement biochemical methods in the study of normal and abnormal bioenergetic and biosynthetic pathways. Since slow freezing rates tend to preserve viability and rapid freezing rates tend to be destructive: a the excellent visualization of enzyme activity by rapid freezing rates presents questions that deserve attention and are discussed elsewhere.3* ’ It would appear that active sites become unmasked by rapid freezing and it will be interesting to probe further into the possible mechanisms by which this is effected.
OF ENZYME
ACTIVITY
149
REFERENCES 1. Melnick, P. J., and Bullock, W. Histochemical study of breast neoplasms. Am. J. Pathol., 35: 706,1959. 2. Melnick, P. J. Histochemical study of lung neoplasms. In Proceedings 21st An&al Reiearch Conference in Pulmonarv Diseases. VA-Armed Forces, 1962, pp. 151-154”. 3. Melnick, P. J. Effect of various freezing rates on the histochemical identification of enzyme activity. Fed. Proc., to be published. 4. Melnick, P. J. Enzyme patterns in tumors demonstrated histochemically in cryostat sections. Annals of the New York Acad. of Sciences, New York, to be published. 5. Meryman, H. T. (Ed). Freezing and drying of bioloaical materials. Annals of the New York Academy of Sciences, New York, 1960. 6. Schiebler, T. H., Pearse, A. G. E., and Wolff, H. H. (Eds). Proceedings 2nd International Congress of Histoand Cytochemistry. Springer-Verlag, Berlin, 1964. 7. Stowell, R. E., Chairman, Conference on comparison of different fixation methods and their significance for histochemistry. In Proceedings 2nd International Congress of Histo- and Cytochemistrv. T. H. Schiebler. A. G. E. Pearse, and H. H. Wolff, Eds., pp. 111-112. SpringerVerlag, Berlin, 1964. 8. Waravdekar, V. S., Goldblatt, P. J., Trump, B. F., Griffin, C. C., and Stowell, R. F. Effect of freezing and thawing on certain nuclear and mitochondrial enzymes of mouse liver. J. Histochem. Cytochem., lb: 498-503, 1964. 9. Wegmann, R. (Ed.). Histochemistry and cytochemistry. In Proceedings 1st International Congress of Histo- and Cytochemistry. Macmillan Co., New York, 1963. ”
I
HISTOCHEMICAL IDENTIFICATION OF ENZYME ACTIVITY IN FROZEN TISSUES AND TUMORS” t P. J. MELNICK University
of California Medical Center, San Francisco; and Veterans Administration Hospital, Martinez, California
For those who work with the microscope, histochemical techniques open a large and fascinating area of research, present exceptional opportunities for a deeper understanding of disease, and are beginning to offer a small but useful number of practical diagnostic aids. The First’ International Congress of Histochemistry and Cytochemistry was held in 1960, and the Second in 1964, marking the recognition of a young specialty whose active growth began scarcely 25 years ago.‘. ’ Its basic principles had, however, been announced more than a century ago by Raspail. The techniques of histochemistry consist of chemical reactions produced in tissue sections, whose visible end-products seen through the microscope, are morphological expressions of the chemical make-up of cells, and of the function of t,heir enzymes. The requirements of a histochemical technique are the following: The method should be specific for the substance or enzyme identified; an easily visible, preferably colored, material or precipitate should be formed so that it is clearly seen through the microscope; and the chemical reaction should be very rapid so that no diffusion occurs and good localization results. The recent rapid expansion of histochemistry justifies a word of caution and conservatism. It will probably be a long time before standard conditions will be generally agreed upon for most histochemical procedures because the chemical react’ions carried out in the tissues are oft’en subtle, and occur in close proximity to many variable and reactive structures in the cells. Controls of several kinds are necessary. * Presented at the First Annual Meeting, Society for Cryobiology, August 24-26, 1964, Washington, D. C. ‘r Aided by U. S. Public Health Service Research Grant CA-07468, and a Veterans Administration Research appropriation. The generous cooperation of the Linde Company is gratefullv acknowledged, as well as the technical assistance of Pauline Heizer. Ph.D.. and the late Carl W. Howell.
False negative reactions may occur when chemical substances are masked or are bound in nonreactive form, when enzymes are held in an inactive state, or when tissues are not properly prepared for the procedure. An example of the latter is loss of soluble enzymes into the incubating solution. False positive reactions are not uncommon because of nonspecific chemical reactions, to enzymes acting on intrinsic unknown substrates, to nonenzymatic reactions of indicators, or to nonoptimal freezing rates of the tissues. Quantitation in histochemistry is very difficult; a feebly active enzyme may appear to be intensely active if it is permitted a long incubation period and thereby a large amount of end-product accumulates in the cells. There is need to check histochemical methods with appropriate biochemical, biophysical, or immunological techniques. There is also need to make comparative studies of the human with a variety of animal species. Most histochemical techniques were developed on normal animal tissues, and the optimal conditions for human tissues may often be different’. This is especially significant for enzymes, because the action of enzymes depends so largely on their precise genet,ically determined structure. Additional variables contributed by disease processes will need to be evaluated in the future. Despite all these complexities, a well defined amount of histochemical knowledge has accumulat,ed that has so far remained intact. In t’he past, histological methods have demonstrated cells merely by coloring them, but this gave only very general notions of the chemical makeup of the structures that were stained, such as basophilia of nuclei or acidophilia of cytoplasm. Today’s histochemical methods are being developed with a view toward demonstrating more precisely the chemical nature of the various structures within cells and of cell products. Methods have been devised to demonstrate various enzymes, carbohydrates, proteins, pigments, nucleic acids, inorganic substances, and
140
:HISTOCHEMICdL
IDENTIFICBTION
lipids. Since histochemistry and cryobiology are both young specialties, we are presented with many problems that require our careful attention.
ENZYMES One of the most exciting developments is the morphological demonstration of enzyme activity within cells. Enzymes are not “living” molecules; they are chemically active protein molecules that depend for their specific activity on their precise structure which is complementary to the structure of the substrates on which they act. If they become denat,ured to such an extent that the structure of their active sites is seriously changed, one rnay still be left with protein molecules, but they are no longer enzymes. Therefore, if ti,ssues can be so processed that their active sites are intact, enzyme activity is often retained. When sections of such tissues are placed into a solution containing a substrate, the specific enzyme, if present, may carry out its chemical action on the substrate if the proper conditions are present, such as pH, temperature, activating ions, and absence of inhibitors. If there is present in the solut,ion a substance with which a product of the reaction will combine to form an insoluble reaction product that can be easily seen with the microscope, the basis of a histochemical t,echnique is present. Most of t,he hydrolases are fairly sturdy and can withstand procedures such as formalin fixation at 5°C. In contrast to the hydrolytic enzymes, most of the dehydrogenases are very unstable in t,issues and will not withstand these procedures, the-refore they are generally studied only in fresh-frozen sections. Freeze-substitution methods are also useful, and freeze-dry methods, although elaborate, may also become practical in the fut’ure. Development, of a histochemical technique for alkaline phosphatase by Gomori in 1939 (and independently by Takamatsu) furnished a strong impetus for the rapid growth of enzyme histochemistry, indeed, for all of histochemistry. Gomori’s technique was the by now well known metal-salt meth.od, and was rapidly followed by similar methods for ot’her phosphatases such as 5’-nucleotidase, glucose-6 acid phosphatase, phosphatase, phosphamidase, and adenosine triphosphatase, for which Gomori’s principle has furnished the basis. The field was greatly ex-
OF ENZYME
ACTIVITY
141
panded when extensive use began to be made of naphtholic substrates for the hydrolytic enzymes. When the enzyme splits off the specific substit’uent from a naphtholic substrate, free naphthol is present. If a diazonium salt has been added to the incubating solution it, couples with the naphthol to form an azo dye that can be easily seen with the microscope as a colored precipit’ate. Today, naphtholic substrates have b een synthesized to demonst’rate very many hydrolaees. Postcoupling and other methods have been devised for certain enzymes, but for most hydrolases t’he simultaneous capture methods with the use of naphtholic substrates are the most widely used. A similar great advance was made in the histochemistry of t’he dehydrogenases by the use of tetrazolium salts. Specific dehydrogenases transfer electrons from their specific substrates, i.e., lactic acid, glutamic acid, etc., to diphosphopyridine nucleotide (DPN) or triphosphopyridine nucleot,ide (TPN) in most cases. In living cells, diaphorases transfer the electrons from the reduced DPN or TPN to the cytochrome chain and thence to molecular oxygen; and the energy released thereby becomes available chiefly for oxidative phosphorylation, i.e., for synthesis of that versatile molecule adenosine triphosphate (-4TP) from adenosine diphosphate ($DP). In t’he histochemical procedure the electrons are, instead, diverted from the reduced DPX or TP?; to a colorless tetrazolium salt added to the solution, which becomes reduced to an insoluble, intensely colored precipitate of formazan. Sometimes respiratory inhibitors such as cyanide are added to t)he solution to inhibit the cytochromes, to ensure that only the tetrazolium is active as an electron acceptor. Sometimes adjuvants such as phenazine methosulfate are added to facilitate the diversion of the electron flow to the tetrazolium, from such enzymes as euccinic dehydrogenase. Tetrazolium salts for dehydrogenase histochemistry have been greatly improved in recent years with the synthesis of nitro-blue tetrazolium and related compounds. The oxidases such as cptochrome oxidase, peroxidase, tyrosinasc system, and monoamine oxidase include a small miscellaneous group whose techniques are gradually being perfected. Histochemistry may become an effective means of studying neoplasme, because t’he great
142
P. J. MELNICK
genetic variability of tumors is reflected in their variable enzyme patterns. It is conceivable that the time may come when surgeons, upon submitting biopsies of malignant tumors to pathologists, will request not only the histological diagnosis, but also a histochemical study of the enzyme patterns, t,o help select the indicated chemotherapy. Similarly, studies of the enzyme patterns of chronic granulomas and other inflammations may some day perhaps add to the effectiveness of antimicrobial therapy. PLAN
OF STUDY
Between 1958 and 1961 a group of about 25 breast cancers and 31 lung cancers were studied with histochemical enzyme methods for 12 hydrolases and 14 oxidative enzymes.‘.’ The results of these studies were reported in detail elsewhere.3’ 4 The outstanding findings consisted, essentially, of enzyme profiles in the differentiated cancers that tended to resemble those of their normal t,issue cells of origin. The undifferentiated cancers, however, presented man) enzyme defects; and t,he more undifferentiated the cell type the fewer enzymes they contained, much like embryonal tissue. Of special interest were the many enzyme delet,ions encountered among the dehydrogenases, since these are so important in the bioenergetic pathways for furnishing controlled chemical energy upon which all life depends. Dehydrogenases must be examined in fresh frozen sections of the tissues, since they do not withstand such procedures as formalin fixation, and even TABLE CODE FOR IDENTIFIC~~TION TO WHICH CODE:
1. 2. 3. 4. 5. 6.
MATERIALS
AXD
METHODS
In 1962, a comparison was begun of the effect of various freezing rates on t’he histochemical identification of enzyme activity. Through the generous cooperat,ion of Dr. Arthus P. Rinfret of the Linde Company, under whose direction the various tissues were prepared and frozen, and shipped in a liquid nitrogen refrigerator to the author’s laboratory, 48 specimens of normal guinea pig liver and kidney were examined. Table 1 contains the code that identifies the tissues and the freezing modalities. Tables 2 and 3 present a compilation of the findings extracted from the large amount’ of detail, for 9 hydrolases and 8 oxidative enzymes. For the purposes of this comparative study, it was decided to use cryost,at sect,ions for bot,h the hydrolases as well as the dehydrogenases. The frozen sections are excellent for both groups of enzymes. In addition, during the past t#hree years, 1
OF NORM.~L GUINEA PIG KIDNEY AND LIVER AND THE CRYOGENIC THEY WER.E EXPOSED (COURTESY OF THE LINDE COMPANY)
MODALITIES
Describes sample type and cooling regime, as follows:
refers to sample type Kidney, l-mm slices Kidney, 2-mm slices Kidney, chunks (3 to 5 mm) Kidney, whole Liver, 2-mm slices Liver, chunks and whole lobes
NUMBER:
a few hours at room temperature will often inactivate them. As was mentioned above, nonoptimal freezing rates present one of the problems that require att’ention in the further development of histochemistry. It seemed possible, therefore, that inactivation of enzymes, by nonoptimal freezing rates in t,he course of making cryostat sections for the study of the dehydrogenases, was t’he basis for at least some of the enzyme deletions found. Problems such as these are increasingly occupying the attent,ion of histochemists.‘, *
refers to cooling regime A. Immersion in liquid Ns(-196°C) B. Immersion in Dry Ice-ethanol (SD-l) at -78”C, followed by immersion in liquid N, C. Immersion in Dry Ice-ethanol (SD-l) at -78”C, storage for one week in dry ice at -78”C, followed by immersion in liquid Nz D. Immersion in Ucon 12 (CClzF,) at -3O”C, followed by immersion in liquid Nz E. Immersion in Ucon 12 at -30°C storage for one week at -25°C (freezer), followed by immersion in liquid Nz F. Cooling to 50°C at approximately 1°C per minute in controlled rate freezer (BF-3), followed by immersion in liquid NB
LETTER:
HISTOCHEMICAL
IDENTIFICATIOX
OF ENZYME
TABLE NORMAL
GLXNE.I
PIG
KIDNEY
143
ACTIVITY
2
AND
LIVER
(CODE
IN
TABLE
1)
Hydrolases
:sterase
Cholinesterase
;lucose-6 Pa%2
TPase
-Gluuronidase I-
Liquid nitrogen (-196°C) 1A 2A 3A 4A 5A 6A Dry Ice-ethanol (-78°C) followed by liquid nitrogen 1B 2B 3B 4B 5B 6B Dry Ice-ethanol (-78°C) 1c 2c 3c 4c 5c 6C “Ucon” (-30°C) followed by liquid nitrogen ID 2D 3D 4D 5D BD “Ucon” (-30°C’ /) IE 2E 3E 4E 5E 6E Slow freeze at -1°C per minute 1F 2F 3F 4F 5F 6F
: + :zk
+
: f+ f+ T + f + f T + f f+ f f f 0 f 0
0 0 0 0 0 0
+ +
: f
z
+ f
1
+
+ +
: 0
z
+ 0
+
1
:
+
+ z 1 0
+
:
:
+
+
+ +
: f
+ +
f *
z
f 0
z
0
:
f
0 0
z
0 0
:
0 0
many examples of a number of normal human tissues obtained at autopsy or from surgical specimens, were similarly examined. Tables 4 and 5 summarize the findings in this mat’erial. The details of the histological techniques used in these studies have been reported in two other communications that describe chiefly the effect’
:
of various freezing rates on histochemical demon&ration of enzymes in tumors.3. 4 Essentially the same principles and findings apply to the tumor material, and the preliminary study of the normal tissues therefore furnished a valuable orientation for the further work on the tumors.
144
P. J. MELNICK TABLE NORMAL GUINEA PIG KIDNEY
3
AND LIVER
(CODS IN TABLE 1) Dehydrogenases
ic
Liquid nitrogen (-196°C) 1A 2A 3A 4A 5A 6A Dry Ice-ethanol (-78°C) followed by liquid nitrogen 1B 2B 3B 4B 5B 6B Dry Ice-ethanol (-78°C) 1c 2c 3c 4c 5c 6C “Ucon” (-30°C) followed by liquid nitrogen ID 2D 3D 41) 5D 6D “Ucon” (-30°C) IE 2E 3E 4E 5E GE Slow freeze at -1°C per minute 1F 2F 3F 4F 5F 6F RESULTS
During the past three years, following the preliminary study on the normal animal and human tissues mentioned above, 140 human tumors were studied in the following manner. Each tumor was cut into several aliquot blocks averaging about 4 mm thick. Several blocks
3lutamic Acid
>
G
Cytochrome Oxidase
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + : 0 0 0 f 0 f f f : : z : + +
were used for other purposes including the effect of fixation, examinat.ion for lipids and for glycogen, etc., but, for the purpose of this cryogenic study, first, one block was frozen rapidly by quenching it in isopentane cooled to -160°C by liquid nitrogen; second, one block was frozen at the rate of -1°C per minute in a
FIGS. 1 to 4 skeletal muscle, rapidly frozen. Cytochrome oxidase, Burstone method. x300. Activity is feeble, and there are scattered focal aggregates of cytochrome c activit,y, probably as a result of crystallized intrinsic cytochrome c due to freezing. FIG. 2. Normal skelet,al muscle, rapidly frozen. Cytochrome oxidase, Burstone method, with added cytochrome c. X300. Activity is intense and uniform. FIG. 3. Normal liver, rapidly frozen. Cytochrome oxidase, Burstone method. X150. Actil-ity is feeble, and there are aggregates of cytochrome c activity, probably as a result of crystallized cytochrome c probably separated out as a result of freezing. FIG. 4. Normal liver, rapidly frozen. Cytochrome oxidase, Burstone method, with added cytochrome c. Sctivity is uniform throughout.
FIG. 1. Normal
145
FIGS. 5 to 8 FIG. 5. Sormal jejunum, rapidly frozen. Cytochrome oxidase, Burstone method. Activity is essentially nonexistent, and is represented by the focal aggregates described above. FIG. 6. Normal jejunum, rapidly frozen. Cytochrome oxidase, Burstone method, with added cytochrome c. Although the focal aggregates of activity persist in this tissue that was quenched at -160°C in isopentane, enzyme activity is seen also in the jejunal epithelium. FIG. 7. Metastasis of carcinoma of the pancreas to the liver, slowly frozen. Succinic dehydrogenase. ~175. Ice crystal artefact is evident. Although activity is seen in a number of the liver cell cords, no enzyme activity is evident in the tumor. FIG. 8. Metastasis of a carcinoma of the pancreas to the liver, rapidly frozen. Succinic dehydrogenase. X17.5. Enzyme activity is seen throughout. This is an example of diminished enzyme activity in slow frozen tissue.
146
HISTOCHEMICAL
IDENTIFICATION
OF ENZYME
ACTIVITY
FIGS. 9 and 10 FIG. 9. Metastasis of an undifferentiated carcinoma of the lung to the liver, slowly frozen. Glutamic dehydrogenase. X125. Although some activity is seen in the liver there is no
enzyme activity in the metastatic tumor. FIG. 10. Metastasis of an undifferentiated carcinoma of the lung to the liver, rapidly frozen. Glutamic dehydrogenase. X125. Although the enzyme activit,y in the liver is enhanced, there is still no activity in the metastatic tumor, an example of a true enzyme deletion. controlled rate Linde BF3 liquid nitrogen freezer; and third, a third block was frozen on a cryostat chuck, whose freezing rate measured with a therm&or varies from -7” to -12°C per minute. Cryostat sections were cut from each block and stored in a deep freeze at -30°C until processed, usually within less than one month. Sections from all three blocks were incubated under identical conditions in the same container, the same incubating solution, and the same length of time. Reference to the reports of this study”, 4 will indicate that when enzyme activity in a given tumor was intense (such as acid phosphat,ase in prostatic cancers), no appreciable difference could be det’ected in the histochemical enzyme product at the three different freezing rat,es. The above described finding should be kept in mind in the interpret,ation of the tables that contain the data on the effect of various freezing rates on normal animal tissues, which generall:,
contain intact, intensely active enzymes, and which would not be expected to have enzyme deletions. As can be seen from the tables, for most of the enzymes strong activity was seen at all freezing rates. However, two types of findings are seen that encouraged us to continue this comparative cryogenic study in tumors, which might be expect’ed to have occasional weakly active enzymes in which differences might be accentuated by different freezing rates, and which also might be expected to have enzyme delet,ions. These two interesting findings in t’he normal tissues are as follows: first, slow freezing tended to diminish or to eliminate activity of the esterases, cholinesternses, certain lipases, and acid and alkaline phosphatnses, whereas rapid freezing preserved their full range of activity int,act ; second, rapid freezing generally abolished, or almost completely abolished, cytochrome c oxidase activity, whereas slow freezing
TABLE 4 CRYOENZYMOLOGY OF NORMAL HUMAN TISSUES
-
Code*
Hydrolases
-
-
Cholin :sterasce 1Lipase
E
A
.-
S~ucleo-
‘?2
A TPase
tidase
2+ 2+
2-t 2+
4+ 4f
3+ 3+
4f 4+
4+
A B
4+ 4+
2+ 2+
3+ 3+
0
4+ 4+
A B
4f 4+
3t 3+
0 0
2+
Skeletal IIlUS&
A B
3-k 3+
2+ 2+
0 0
Brain
A B
2+
1+
4+
3+
A B
2+ 4-t
A B A B
Myocardium
Nerve Pancreas Jejunum
Sulfatase
.-
4+ 4f
Liver
cumoidase
-
eucim :P tlanim AmiAminax nase
--
A B
Kidney
/B-GlU
4+
4+ 4-k
3f 3+
2+ 2+
4+ 4+
4+ 4+
4+ 4+
4+ 4+
4+ 4+
3+ 3f
4+ 4+
1+ 1+
4-t 4+
4+ 4t
1+ 4+
3+ 3+
1+ 1+
4+ 4+
2+
1+ If
2+ 2+
4+ 4+
1+ 1+
2+ 2+
1+ 1+
4+ 4+
0 1+
2+ 2+
2+ 3+
4+ 4+
If
it
2+
1+ 1+
1+ If
4+ 4+
1+ 3+
0 1+
2+ 2+
0 1+
4f 4+
2+
t:
4+
1+ 1+
1+ 1+
2:
4+ 4f
3+ 3+
3f 3+
3+ 3+
3+ 3+
4f 4f
4+ 4+
3+ 3f
2+ 4+
2+ 2+
4+ 4+
4+ 4+
3+ 3+
4+
4+ 4+
3+ 3+
4+ 4f
4+ 4+
2+ 2+
1+ 4+
1+
2+
4+ 4+
* Code: A = slow fr’ eeze;
w 4+ If
I 4+ I
5 = rapid
freeze.
-
-
-
TABLE 5 CRYOENZYMOLOGY OF NORMAL HUMAN TISSUES Dehydrogenases
Code*
suc-
SUCchic
2+
-
30 74;
cink system
IScitric
Glutami
GluEth: I- coseml I 6P
I acti c
coni
“GE-$8
2” g.2 20 u
2% es” 26 k” u
A B
4+ 4+
2+
4+ 4f
4+ 4+
I: 2
2
3+
3t 3+
2+ 2+
0 0
4+ 4f
4+ 4t
3t 0
4+ 4f
A B
4+ 4+
3+ 3+
4f 4+
4f 4t
3+ 3+
3+ 3t
4+ 4+
4+ 4+
4f 4+
3+ 4+
1-t
w
4+ 4+
4+ 4+
2+ i
4+ 4+
A B
4+ 4f
2+
4+ 4+
4+ 4+
3+ 3+
3+ 3+
2+
2+
4+
3+
4+ 4+
1-t 3+
0 0
4+ 4+
4+ 4+
3+ f
4+ 4+
Skeletal muscle
A B
2-t
2+
2+ 4+
1-t 3+
2+ 2+
1+ 3+
1+ 3+
2+
4+ 4t
;I
0 0
4+ 4+
3+ 3+
2+
4+
0
4+ 4+
Brain
A B
1+ 4+
0
2+
3+ 3+
4t 3-t
2+
2+
2+
2+
3+
4+
4+
3+
1+ 3+
0 0
3+ 3+
2+ 2+
3+ f
4+ 4+
A B
1+ 4f
0
1+ 3+
3+ 3+
1-t 3f
1+ 3+
a4+
2+ 3t
1+ 3+
0 0
3+ 3+
1+
4+
2+
3+ i
4+ 4+
Pancreas
A B
4+ 4f
3+ 3+
4+ 4f
4f 4+
4+ 4-f
4+ 4+
4+ 4+
4+ 4+
4+ 4+
4+ 4+
0 0
4+ 4+
4+ 4t
3+ 1+
4f 4+
Jeiunum
A B
3f 3f
2+
2+
2+
2+
4+
4+
4+
4+ 4f
t=
0 0
4+ 4+
4+ 4-k
2+
3+
1+ 3+
2+
4+
0
4+ 4+
Kidney Liver Myocardium
Nerve
4+ 4+ 1- r * Code: A = SIOW Iret
a+ 1+ 2+ 2+
4+
2+
e; I i := ra
2+
1+
-
Id freeze. 148
HISTOCHEMICAL
IDENTIFICATION
rates tended to :retain at least partial activity, especially in the whole livers and kidneys. The reader is referred to the two other reports on other aspects of the cryoenzymology of the 140 tumors studied3# ’ for discussions and principles brought out by this material. Summarized briefly: first, addition of cytochrome c to the incubating solution brought out full activity of the enzyme in all tissues, because freezing apparently crystallizes and sequestrates intrinsic cytochrome c making it unavailable as a substrate; seconsd, in the case of weakly active enzymes, slow freezing rates fail to bring out activity, leading to possible wrong interpretations of enzyme deletions, whereas quenching in isopentane at -160°C brings out the full range of activity; third, by such an approach determinations of true enzyme deletions in tumors can perhaps be est’ablished; and fourth, histochemical enzyme techniques should be able significantly to supplement biochemical methods in the study of normal and abnormal bioenergetic and biosynthetic pathways. Since slow freezing rates tend to preserve viability and rapid freezing rates tend to be destructive: a the excellent visualization of enzyme activity by rapid freezing rates presents questions that deserve attention and are discussed elsewhere.3* ’ It would appear that active sites become unmasked by rapid freezing and it will be interesting to probe further into the possible mechanisms by which this is effected.
OF ENZYME
ACTIVITY
149
REFERENCES 1. Melnick, P. J., and Bullock, W. Histochemical study of breast neoplasms. Am. J. Pathol., 35: 706,1959. 2. Melnick, P. J. Histochemical study of lung neoplasms. In Proceedings 21st An&al Reiearch Conference in Pulmonarv Diseases. VA-Armed Forces, 1962, pp. 151-154”. 3. Melnick, P. J. Effect of various freezing rates on the histochemical identification of enzyme activity. Fed. Proc., to be published. 4. Melnick, P. J. Enzyme patterns in tumors demonstrated histochemically in cryostat sections. Annals of the New York Acad. of Sciences, New York, to be published. 5. Meryman, H. T. (Ed). Freezing and drying of bioloaical materials. Annals of the New York Academy of Sciences, New York, 1960. 6. Schiebler, T. H., Pearse, A. G. E., and Wolff, H. H. (Eds). Proceedings 2nd International Congress of Histoand Cytochemistry. Springer-Verlag, Berlin, 1964. 7. Stowell, R. E., Chairman, Conference on comparison of different fixation methods and their significance for histochemistry. In Proceedings 2nd International Congress of Histo- and Cytochemistrv. T. H. Schiebler. A. G. E. Pearse, and H. H. Wolff, Eds., pp. 111-112. SpringerVerlag, Berlin, 1964. 8. Waravdekar, V. S., Goldblatt, P. J., Trump, B. F., Griffin, C. C., and Stowell, R. F. Effect of freezing and thawing on certain nuclear and mitochondrial enzymes of mouse liver. J. Histochem. Cytochem., lb: 498-503, 1964. 9. Wegmann, R. (Ed.). Histochemistry and cytochemistry. In Proceedings 1st International Congress of Histo- and Cytochemistry. Macmillan Co., New York, 1963. ”
I