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Osmium-zinc iodide reactivity in human blood and bone marrow cells.

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Osmium-Zinc Iodide Reactivity in Human Blood a n d
Bone Marrow Cells
MICHAEL A. CLARK AND G. ADOLPH ACKERMAN
Department of Anatomy, Ohio State University,
Columbus, Ohio 43210
ABSTRACT
Buffy coats from blood and bone marrow were fixed in phosphatebuffered glutaraldehyde, exposed to the osmium-zinc iodide (OZI) reagent for
24 hours, dehydrated and embedded in Epon 812. OZI reactivity of blood and
bone marrow cells was selectively confined to the cisternae of the rough endoplasmic reticulum (RER), Golgi complex, nuclear envelope and to the mitochondrial matrices; membranes and other organelles were non-reactive. Some
variation in intensity and distribution of OZI reactivity was evident within individual cells and organelles. Continuities between the cisternae of the nuclear
envelope and RER as well as between these cisternae and those of the Golgi complex were more conspicuous in OZI preparations than in specimens prepared for
routine electron microscopy.
The amount and distribution of the cisternal elements and mitochondria within the developing leukocytes and erythrocytes of the bone marrow were evaluated
using the OZI technique. All leukocyte granules and their precursor forms fail
to stain with the OZI reagent; portions of the Golgi complex most closely associated with the packaging of the cytoplasmic granules also are non-reactive following exposure to the 021 reagent. Reactivity is absent in mature erythrocytes
while mitochondria and cisternal components of immature erythroid cells yield
positive OZI reactions. Heat, methanol and cyanide inhibit OZI reactivity while
a dimorphism of OZI staining is induced between mitochondria and cisternal
components by N-ethylmaleimide,
The osmium-zinc iodide (OZI) reaction
and its modifications have been applied primarily in the tracing of nerve fibers and
their terminations (Akert and Sandri, '68;
Champy, '13; Cruz, '62; Droz, '58; Maillet,
'63; Stockinger and Graf, '65). In addition,
Stockinger and Graf ('65) reported diffuse
staining of transitional epithelial cells of
the urinary bladder and reticular cells of
the lymph node as well as intracellular
deposition of OZI reaction product within
the rough endoplasmic reticulum (RER) ,
sarcoplasmic reticulum, Golgi complex and
mitochondria of fibroblasts, skeletal muscle, fat cells and plasma cells in the mouse.
Recently major differences between the
intracellular OZI reactivity of the Langerhans cells and the epithelial cells of the
human skin were reported (Niebauer et al.,
'69) and the intracellular process of melanin granule formation in the Langerhans
cells traced by means of this technique.
ANAT. REC., 170: 81-96
Our interest in the OZI reaction was initiated by the fact that the melanin granules
of Langerhans cells and the cytoplasmic
granules of neutrophils are DOPA oxidasepositive and for this reason it was postulated that the OZI reaction might prove
valuable in studies of the developing neutrophils of human bone marrow.
It is the purpose of this investigation to
describe the intracellular localization of
OZI reaction product in the various cells
of the blood and bone marrow of man
since, except for our preliminary observations (Ackerman and Clark, ' 7 0 ) , this
reaction has not been described or evaluated in these tissues.
Received July 29, '70. Accepted Oct. 20, '70.
1 Supported in part by National Institutes of Health
Fellowship 5FOl-GM-39,266-02 ( A P A ) from the National Institute of General Medical Sciences.
2 Supported in part by National Institutes of Health
grant AM-HE-12064-12from the National Institute of
Arthritis and Metabolic Diseases.
81
82
M. A. CLARK AND G. A. ACKERMAN
MATERIALS AND METHODS
RESULTS
Blood and bone marrow specimens obtained from healthy medical students were
anticoagulated with phosphate-buffered
disodium ethylenediamine tetraacetic acid
and buffy coats prepared. Buffy coat pellets
were fixed with 2.5% glutaraldehyde in
0.1 M phosphate buffer, pH 7.4 containing
1% sucrose for two and one-half hours at
4°C. Pellets cut into small strips were incubated in the OZI reagent (Niebauer et
al., '69) for 18-24 hours at 4°C in the dark
and subsequently dehydrated in ethanol
and embedded in Epon 812. In addition,
unfixed pellets and bone marrow fragments were placed directly in the OZI reagent for 24 hours at 4"C, these tissues
showed the same distribution of reaction
product as those prefixed in glutaraldehyde. Glutaraldehyde-fixed material proved
superior in preservation and ultrastructural detail, Thick (0.5 ,-1.O p ) sections
were examined unstained with phase and
bright field microscopy while thin sections
were studied either unstained or after
staining with alcoholic uranyl acetate and
lead citrate with an RCA EMU3f electron
microscope.
Control experiments were performed on
buffy coat slices previously fixed in glutaraldehyde and exposed to the following
agents for one hour at room temperature :
( 1 ) 0.01 M sodium fluoride, ( 2 ) 0.02 M
iodoacetic acid, ( 3 ) 0.02 M sodium azide,
( 4 ) 0.02 M potassium malonate, ( 5 ) 0.05
M 2,4-dinitrophenol, (6) 0.02 M sodium
cyanide, and (7) 70% methanol. Other
controls employed were 0.1 M N-ethylmaleimide in 0.1 M phosphate buffer, pH
7.4 for four hours at 37"C, and the heating
of tissues in 0.1 M phosphate buffer, pH
7.4 and in the glutaraldehyde fixative for
30 minutes in a boiling water bath. Controls were rinsed in distilled water, subjected either to the 021reagent as detailed
above or dehydrated and embedded in
Epon 812 without further treatment. Additional controls consisted of the exposure of
tissue slices to saturated aqueous iodine
and zinc iodide solutions for 18-24 hours
at 4"C, and to 2% aqueous osmium tetroxide adjusted to pH 4.0 with 1 N hydrochloric acid for 48 hours at 37°C.
All cells of the blood and bone marrow
with the exception of mature erythrocytes
exhibit a positive OZI reaction (fig. 1 ) . In
thick sections, staining occurs in the nuclear envelope, Golgi and small strands
present within the cytoplasm of many cells
while the most intense reactivity observed
at the light microscopic level occurs in
mitochondria. Electron microscopy reveals
the sharp localization of the OZ1 reaction
product within the cisternal system of the
cells, viz., the cisternae of the nuclear envelope, Golgi complex and RER (figs. 2, 3 )
and within the matrices of mitochondria
(figs. 4-6). The membranes of these structures, however, are non-reactive (figs. 46 ) and the reaction product is never seen
within cytoplasmic granules, centrioles,
microtubules, nuclear pores or pinocytotic
vesicles. Variations in cisternal and mitochondrial staining may be encountered and
can be associated with the area of the tissue block examined. In addition, intracellular variations in the reactivity of organelles occasionally may be noted within
individual cells (fig. 6). Changes in the
interrelationships of the nuclear envelope,
Golgi complex and RER as well as mitochondrial size and number are reflected
by the OZI reaction. The overall reactivity
of individual cells varies with the stage of
development and with number and distribution of the organelles present. Connections of the nuclear envelope with the Golgi
complex (fig. 3 ) and RER (fig. 5) are
readily observed as are cisternal interconnections and transitional vesicles between
the Golgi complex and RER (fig. 3 ) .
Granulocytes. The OZI reactivity observed in myeloblasts is localized within
the mitochondria and cisternal components
(fig. 7). The Golgi is small and only a few
strands of RER are present within these
cells. The reactive Golgi and RER increase
in extent and complexity during the promyelocyte phase of development. Transitional vesicles are conspicuous and interconnections between the nuclear envelope,
Golgi and RER can be readily visualized in
OZI preparations (fig. 3 ) . Golgi cisternae
and vesicles associated with the final packaging of primary (azurophil) granules,
however, are non-reactive (fig. 8). Primary granules and their developmental
83
OSMIUM-ZINC IODIDE REACTIVITY
forms also are consistently nonreactive.
Mitochondria are numerous in the promyelocytes, exhibit OZI-reactivity and decrease in size during the final phase of
this developmental stage. The OZI reaction
reflects the changes in cisternal components of the neutrophil myelocytes, viz.,
the reduction in size and extent of the
RER and the decrease in the size and prominence of the Golgi complex. Secondary
(specific) granules and their developmental forms as well as a few of the Golgi
cisternae are consistently non-reactive.
The amounts of RER present in the metamyelocytes and mature neutrophils is less
than in the more immature granulocytes
and the reactive RER is often aggregated
into condensed networks. The mitochondria of the mature neutrophils are sparse,
of small size and OZI-positive; the reactive
Golgi is small and condensed. No reactive
granules are seen within either the metamyelocytes or mature neutrophils (fig. 9).
Similar patterns of OZI reactivity are noted
in developing and mature eosinophils and
basophils of the blood and bone marrow;
the cytoplasmic granules of these granulocytes and their precursors are uniformly
non-reactive (fig. 9).
Agranulocytes. The cisternal elements
and mitochondria of lymphocytes and
plasma cells exhibit OZI reactivity; cytoplasmic granules when observed are nonreactive (fig. 10). The mature and immature monocytes display reactive Golgi,
RER, nuclear envelopes and frequent cisternal interconnections (fig. 10). The RER
in these cells is not as dilated as that observed in either the myelocytes or promyelocytes. Mitochondria are numerous and
OZI-positive. The cytoplasmic granules
present in the monocytes and their developmental forms are non-reactive and the
Golgi components associated with granule
formation fail to exhibit OZI reactivity.
Erythroid elements and megakaryocytes.
The small cisternae of the Golgi complex
and the rare strands of RER present in
nucleate erythroid cells are more conspicuous in OZI preparations than in routinely
prepared specimens (figs. 5, 1 1 ) . These
cisternal components as well as the nuclear
envelope and mitochondria exhibit OZI
reactivity while rhopheocytotic vesicles are
non-reactive; connections of the nuclear
envelope with the Golgi complex and RER
have been observed (fig. 5). The mitochondria and cisternal components of
megakaryocytes and platelets stain with
the OZI reagent. The difference between
the demarcation membranes and RER is
apparent since the demarcation membranes are OZI-negative; cytoplasmic granules present within platelets and megakaryocytes are non-reactive.
Methanol and heat completely inhibit
OZI reactivity while the only metabolic inhibitor to produce an overall decrease in
reactivity is cyanide. Fluoride causes an
intensification of the OZI reaction; patches
of diffuse reactivity are observed occasionally within the cytoplasmic matrices after
treatment with fluoride. Iodoacetate and
dinitrophenol produce a diffuse staining of
the cytoplasm and nucleus of the cell; cytoplasmic granules and membranes remain
unstained in these preparations. Mitochondrial reactivity is inhibited by N-ethylmaleimide but cisternal activity is not altered
by this agent. Specimens treated with metabolic inhibitors and not subsequently exposed to OZI reagent showed no staining.
DISCUSSION
The histochemical nature of the OZI reaction is not clear although cellular reactivity has been attributed to certain reducing substances, viz., catecholamines and
ascorbic acid (Stockinger and Graf, '65)
and to lipid moieties unmasked from lipoprotein by the OZI reagent (Niebauer et
al., '69). Although reactivity is prevented
by exposure to lipid solvents such as
methanol, it is unaltered by glutaraldehyde
fixation and can be greatly inhibited by
heat and to a lesser extent by cyanide.
Therefore, if lipid moieties are involved,
heating rather than fixation must be capable of inhibiting the unmasking of these
reactive groups. In addition, the presence
of lipid can be questioned since leukocyte
granules and erythrocytes fail to yield a
positive OZI reaction but do exhibit positive histochemical reactions for lipids and
phospholipids (Ackerman, '64; Bloom and
Wislocki, '50; Lillie and Burtner, ' 5 3 ) . The
strong OZI reaction of the mitochondria1
matrices as contrasted with the low concentration of lipid within this compartment
(Mahler and Cordes, '66) cast further
84
M. A. CLARK AND G . A. ACKERMAN
doubt on the concept of lipid being involved in this reaction. The reactivity of
the cisternal system is not affected by
N-ethylmaleimide but this agent completely inhibits mitochondrial staining.
Compounds containing reactive sulfhydryl
groups could be involved in mitochondrial
OZI reactivity, however, a different mechanism apparently accounts for cisternal
staining. If sulfhydryl-containing compounds are involved in OZI staining, it is
difficult to understand why leukocyte granules, erythrocytes and epithelial cells of the
skin known to be rich in these substances
(Ackerman, '64; Barrnett and Seligman,
'54) fail to yield a positive OZI reaction.
The OZI reaction has been reported to be
independent of glycolytic and respiratory
enzyme activity (Stockinger and Graf, '65;
Jabonero et al., '62). Our observations support this conclusion since dinitrophenol,
iodoacetate, malonate, fluoride and azide
do not inhibit OZI reactivity. Only heat
and cyanide proved capable of reducing or
inhibiting this reaction which indicates
there may be an enzymatic basis for OZI
reactivity in the cells of the blood and bone
marrow. The similarities in the localization
of the OZI reaction product, cholinesterase
and ATPase activities have been compared
although these enzymes have not been
shown to account for this reaction (Stockinger and Graf, '65). A similar pattern of
association of OZI reactivity and the histochemical and biochemical presence of
DOPA oxidase in Langerhans cell granules
and cisternal components (Seiji et al., '63)
is evident although the OZI reaction was
not considered to demonstrate this enzyme
(Niebauer et al., '69). Leukocyte (neutrophi1 and eosinophil) granules as well as
erythrocytes exhibit a positive histochemical reaction for DOPA oxidase activity although this reaction in blood cells may be
related to or initiated by a peroxidase (van
Duijn, '57). Although primary granules in
neutrophils are reactive for DOPA oxidase
and myeloperoxidase, differences in the
intracellular distribution of these enzyme
activities in neutrophils has been indicated
(Kubo, '67). The possibility of the OZI
reactive substance being attributable to
either DOPA oxidase or related to myeloperoxidase can be dispelled since the leukocyte granules are OZI-negative.
Since the OZI reaction product is localized specifically in the nuclear envelope,
RER, Golgi and mitochondria of blood and
bone marrow cells, this procedure has
proven valuable in assessing the extent
and interrelationships of the cisternal components as well as studying changes in the
size and number of mitochondria during
blood cell development and maturation.
Although these changes are recognized by
routine electron microscopy, they are more
readily appraised in OZI preparations. In
relation to the concept of granulogenesis
occurring in developing neutrophils and
eosinophils, the OZI reaction represents a
significant departure from the data obtained by the peroxidase and acid phosphatase procedures. The entire canalicular
system, the packaging, condensation and
maturation of the primary neutrophil
(Ackerman, '64; Bainton and Farquhar,
'68a,b; Dunn et al., '68; Kubo, '67; Wetzel,
'70a,b) and eosinophil (Bainton and Farquhar, '70; Breton-Gorius and Guichard,
'69; Miller and Herzog, '69) granules can
be followed by means of enzymatic procedures, however, OZI reactivity disappears
within the Golgi cisternae and condensing vacuoles associated with the packaging
of these OZI-negative granules. These data
indicate that a significant alteration of the
OZI reactant must occur at the point of
granule condensation either by an irreversible binding of the substance or a loss
of reactive groups. Alternatively, the OZI
reactive substance might remain within
the cisternae and not be transported into
the developing granules. The possibility of
the direct transformation of the dilated
cisternae of the RER into primary granules
in early promyelocytes in man (Scott and
Horn, '70) is not confirmed in OZI preparations.
ACKNOWLEDGMENTS
Appreciation is expressed to Barbara
Jordan and Anita Topson for their technical assistance during this investigation and
to Dr. Arthur Sagone who performed the
bone marrow aspirations.
LITERATURE CITED
Ackerman, G. A. 1964 Histochemical differentiation during neutrophil development and maturation. Ann. N. Y. Acad. Sci., 113: 537-565.
OSMIUM-ZINC IODIDE REACTIVITY
__-
1968 Ultrastructure and cytochemistry
of the developing neutrophil. Lab. Invest., 19:
290-302.
Ackerman, G . A., and M. A. Clark 1970 Ultrastructural cytochemistry of human blood and
bone marrow cells examined by the osmiumzinc iodide method. Anat. Rec., 166: 269.
Akert, K., and C. Sandri 1968 An electron-microscopic study of zinc iodide-osmium impregnation of neurons. t. Staining of synaptic vesicles at cholinergic junctions. Brain Res., 7:
286-295.
Bainton, D. F., and M. G. Farquhar 19681 Differences i n enzyme content of azuraphil and
specific granules of polymorphonuclear leukocytes. I. Histochemical staining of bone marrow cells. J. Cell Biol., 39: 286-298.
196813 Differences in enzyme content
of azurophil and specific granules of polymorphonuclear leukocytes. 11. Cytochemistry and
electron microscopy of bone marrow cells. J.
Cell Biol., 39: 299-317.
1970 Segregation and packaging of
grznule enzymes in eosinophilic leukocytes. J.
Cell Biol., 45: 54-73.
Barrnett, R. J., 2nd A. M. Seligman 1954 Histochemical demonstration of sulfhydryl and disulfide groups of protein. J. Nat. Cancer Inst.,
14' 769-803.
Bloom, M. L., and G. B. Wislocki 1950 The
localization of lipids i n human blood and bone
marrow cells. Blood, 5: 79-88.
Breton-Gorius, J., and C. Guiehard 1969 Etude
au microscope electronique de la localisation
des peroxydases dans les cellules de la moelle
oeseuse humaine. Nouv. Rev. Franc. Hemat.,
9: 678-687.
Champy, C. 1913 Granules et substances
rkduisant l'iodure d'osmium. J. d'Anat. Physio.,
4: 323-343.
Cruz, A. R. 1962 Histochemical significance of
the osmium-iodide method for autonomic
nerves. Acta Anat., 49: 232-235.
Droz, B. 1958 Eine histochemische Methode
zur Darstellung der vegetativen Innervation
der Haut. Acta Neuroveg., 18: 311-319.
Dunn, W. B., J. H. Hardin and S. S. Spicer 1968
Ultrastructural localization of myeloperoxidase
in human neutrophil and rabbit heterophil and
eosinophil leukocytes, Blood, 32: 935-944.
Jabonero, V., M. T . Genis and L. Santos 1962
Beobactungen uher die osmium-zink-jodid-
85
affinen E l e m a t e der Vorstdherdriise. Z. mikr.
Anat. Forsch., 69: 167-190.
Kubo, M. 1967 A cytochemical electron microscopic study of granulogenesis in neutrophils.
Peroxidase and DOPA-oxidase reactions. Nagoya J. med. Sci., 30: 327-340.
Lillie, R. D., and R. J. Burtner 1953 Stable
sudanophilia of human neutrophil leucocytes in
relation to peroxidase and oxidase. 3. Histochem. Cytochem., 1: 8-26.
Mahler, F., and E. H. Cordes 1966 Biological
Chemistry, Harper and Row, New York.
Maillet, M. 1963 Le rCactif au tetraoxyde
d'osmium-iodure du zinc. 2. mikr. Anat.
Forsch., 70: 397425.
Miller, F., and V. Herzog 1969 Die Lokalisation von Peroxydase und saurer Phosphatase in
eosinophilen Leukocyten wahrend der Reifung.
Elekronenmikroskopisch-cytochemische Untersuchungen 2m Knochenmark von Ratte und
Kaninchen. Z. Zellforsch., 97: 84-110.
Niebauer, G., W. S. Krawczyk, R. L. Kidd and G.
F. Wilgram 1969 Osmium zinc iodide reactive sites in the epidermal Lan,gerhans cell. J.
Cell Biol., 43: 80-89.
Scott, R. E., and R. G. Horn 1970 Ultrastructural aspects of neutrophil granulocyte development in humans. Lab. Invest., 23: 202-215.
Seiji, M., K. Shimao, M. S. C. Birbeck and T. B.
Fitzpatrick 1963 Subcellular localization of
melanin biosynthesis. Ann. N. Y. Acad. Sci.,
100: 497-547.
Stockinger, L., and J. Graf 19'65 Elektronenmikroskopische Analyse der Osmium-Zinkjodid
Methode. Mikroskopie, 20: 16-35.
vanDuijn, P. 1957 Histochemistry of DOPA
factors. 111. Inactivation experiments on the
DOPA factors in neutrophilic and eosinophilic
leucocytes and erythrocytes. Acta Physiol.
Pharmacol. Neer., 5 : 428-444.
Wetzel, B. K. 1970a The fine structure and cytochemistry of developing granulocytes, with
special reference to the rabbit. In: Regulation
of Hematopoiesis. A. S. Gordon, ed. AppletonCentury-Crofts, New York, pp. 769-817.
1970b The comparative fine stru'cture
of normal and diseased mammalian granulocytes. In: Regulation of Hematopoiesis. A. S .
Gordcn, ed. Appleton-Century-Crofts, New York,
PP. 819-872.
Abbreviations
B, myeloblast
C, early neutrophilic
promyelocyte
D, late neutrophilic
promyelocyte
E, eosinophilic myelocyte
L, lymphocyte
M, monocyte
N, mature neutrophil
P, promyelocyte
R, developing red blood
cells
a, cytoplasmic granules
c, centriole
d, mitochondria1 cristae
e, nuclear envelope
g, Golgi complex
i, immature granule forms
m, mitochondria
p. nuclear pore
r, rough endoplasmic
reticulum
t, transitional vesicles
v, rhopheocytotic vesicle
Bone marrow buffy coats were fixed i n phosphate-buffered glutaraldehyde and exposed to the 021 reagent for 18-24 hours. Unless otherwise
noted, all thin sections were counterstained with uranyl acetate and lead
citrate.
PLATE 1
EXPLANATION O F FIGURES
86
1
The general topography of the marrow following the OZI reaction is
evident in this preparation. Reactivity is observed in the nuclear
envelope, in a number of dark cytoplasmic granules which represent
mitochondria ( m ) and i n a scattering of poorly-defined cytoplasmic
strands and granules. Bright-field microscopy, no counterstain. X 800.
2
The cisternae of the nuclear envelope ( e ) , Golgi complex ( 9 ) and
rough endoplasmic reticulum ( R E R ) [I] i n this neutroplhilic myelocyte are 021-positive. Reaction product is conspicuously absent from
the cytoplasmic granules ( a ) and some regions of the Golgi complex.
No counterstain. X 36,000.
3
Strands of RER ( r ) are seen in continuity with the nuclear envelope
( e ) and transitional vesicles ( t ) appear between the nuclear envelope and Golgi complex ( g ) . Only traces of reactivity are present
within the mitochondria (m) while the immature granules ( i ) and
nuclear pore ( p ) in this neutrophilic promyelocyte are unstained.
X 54,000.
OSMIUM-ZINC IODIDE REACTIVITY
Michael A. Clark and G. Adolph Ackerman
PLATE 1
a7
PLATE 2
EXPLANATION O F FIGURES
88
4
Reactivity is intense within the matrices of the mitochondria ( m ) in
this lymphocyte. Note that the mitochondrial membranes and outer
compartment (arrows) as well as membranes of the cristae and the
intercristal space ( d ) fail to stain. The nuclear envelope ( e ) and a
strand of RER ( r ) are reactive and the nuclear pore ( p ) is unstained.
X 75,000.
5
Rough endoplasmic reticulum (r) is in close proximity or continuity
(arrows) with the nuclear envelope (el of a normoblast. Reactivity also
appears in the mitochondria (m), but is absent from the rhopheocytotic
vesicles (v). x 22,000.
6
Note the marked variations in mitochondrial staining within a single
cell. Only the mitochondria1 matrices are reactive while the intercristal space ( d ) and outer compartments (arrows) do not stain.
X 67,000.
OSMIUM-ZINC IODIDE REACTIVITY
Michael A. Clark and G . Adolph Ackerman
PLATE 2
89
PLATE 3
EXPLANATION OF FIGURE
7
90
Several stages of neutrophil development are shown. In the myeloblast (B) scant amounts of RER ( r ) and few mitochondria are seen
and the nuclear envelope ( e ) is reactive; a strand of RER is in continuity with the nuclear envelope (arrow). When primary granulogenesis is occurring i n the early neutrophilic promyelocyte ( C ) the
cisternae of the RER ( r ) become dilated but retain their reactivity and
the cytoplasm contains non-reactive primary granules ( a ) . Several
mitochondria (in) are apparent and i n the cell illustrated, reactivity
is absent from the n u d e a r envelope ( e ) . In the late neutmphil promyelocyte ( D ) the cisternlae of the RER ( r ) are less distended than i n
the early promyelocyte and mitochondria ( m ) are decreased in size
and number. In this cell, reactivity appears in the nuclear envelope
( e ) and Golgi complex ( g ) . Note that n o reactivity is observed in
mature granules ( a ) or in immature granule forms ( i ) i n close psoximity with the Golgi complex. x 12,000.
OSMIUM-ZINC IODIDE REACTIVITY
Michael A. Clark and G. Adolph Ackerman
PLATE 3
91
PLATE 4
EXPLANATION O F F I G U R E S
92
8
In the late neutrophilic promyelocyte, cisternae of RER (I)
the,nuclear envelope ( e ) and central region of the Golgi complex ( 9 ) and
mitochondria ( m ) stain while some portions of the Golgi (arrow) are
non-reactive. Transitional vesicles ( t ) between the Golgi and RER
are seen but reactivity is not observed i n the mature primary granules
( a ) or the centriole ( c ) . X 31,000.
9
The Golgi complex ( 9 ) and RER ( r ) are clumped in these mature
neutrophils ( N ) and n o mitochondria are visible. Granules (a) i n the
eosinophilic myelocyte ( E ) do not stain with OZI reagent while the
nuclear envelope ( e ) and dilated cisternae of RER ( r ) are reactive.
Note the variation in mitochondria1 steining ( m ) . x 11,000.
OSMIUM-ZINC IODIDE REACTIVITY
Michael A. Clark and G . Adolph Ackerman
PLATE 4
93
PLATE 5
EXPLANATION O F FIGURES
10 OZI reaction product appears in the nuclear envelope, scattered
strands of RER ( r ) , mitochondria and Golgi complex ( 9 ) of three
1ymFhocytes ( L ) . In the developing monocyte ( M ) , extensive RER
( r ) , mitochondria and the nuclear envelope are displayed by the OZI
reaction but the cytoplasmic granules ( a ) do not stain. Note nonreactive developing granules (arrows) in the vicinity of the Golgi
complex ( g ) . x 8,800.
11
94
Reactivity is apparent in the RER ( r ) and mitochondria ( m ) of this
promyelocyte ( P ) , but absent from the nuclear envelope and granules
( a ) . In developing red blood cells ( R ) reactivity is seen in the nuclear envelope ( e ) , Golgi complex ( g ) and some mitochondria (m).
Note the mitotic figure at the lower left where reactivity is seen in
the remnants of the nuclear envelope ( e ) . In the nucleate red cell
a t the upper right it is of interest to note a non-reactive mitochondrion (m). x 7,300.
OSMIUM-ZINC IODIDE REACTIVITY
Michael A. Clark and G. Adolph Ackerman
PLATE 5
95
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