Sulfated glycosaminoglycans in guinea pig basophils studied by means of cationic colloidal gold

 Bone marrow embedding in the hydrophilic resin, Lowicryl K4M, followed by cationic colloidal gold (CCG, pH 1.0) staining was used to study the sulfated glycosaminoglycans (GAGs) and their sites of sulfation ultrastructurally in various maturational stages of both basophil granulocytes and basophil granules in the guinea pig. CCG at pH 1.0 is specific for sulfated GAG staining. Basophil granulocytes and granules reacted positively to CCG with a variety of staining according to the stage of maturation. The formation of basophil granules takes place throughout the myelocyte stage. Early basophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late myelocytes contain a small and less active Golgi apparatus as judged by CCG staining. All the immature granules and some of the granules with characteristic ultrastructure stained positively. However, some of the mature granules had lost their affinity for CCG upon maturation. Interestingly, strongly positive CCG staining was also observed in the trans to transmost Golgi apparatus. This indicates that sulfation of GAGs occurs in the trans to transmost Golgi apparatus in all maturational stages of basophil granulocytes. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG staining. Accepted: 17 July 1997     

Sulphated Glycosaminoglycans in Guinea Pig Eosinophils Studied by Means of Cationic Colloidal Gold

Using bone marrow embedded in hydrophilic resin Lowicryl K4M and cationic colloidal gold pH 1.0 labelling, we studied sites of sulphation and sulphated glycosaminoglycans ultrastructurally in various maturational stages of both eosinophil granulocytes and eosinophil granules of guinea pig. Eosinophil granules reacted positively to cationic gold, the pattern of labelling varying according to the degree of cell maturation. The formation of eosinophil granules takes place throughout the myelocyte stage. Early eosinophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late ones contain a small and less active Golgi apparatus. All the immature granules were labelled positively. However, mature granules with a central crystal bar lost their affinity towards colloidal gold. Interestingly, strong colloidal gold labelling was also observed in the trans to transmost Golgi apparatus, especially in immature eosinophil granulocytes. This indicates that sulphation of glycosaminoglycans occurs in the trans to transmost Golgi apparatus of eosinophil granulocytes. Prior absorption with poly-L-lysine prevented colloidal gold labelling of tissue sections. Methylation of sections at 37°C did not alter the gold labelling, whereas the labelling disappeared after methylation at 60°C. Prior treatment with chondroitinase ABC or heparinase I abolished the majority of colloidal gold labelling in immature eosinophil granules. Taking these results together, we conclude that immature eosinophil granules contain sulphated glycosaminoglycans including chondroitin sulphate or heparan sulphate or both.     

Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach

Insulin and C-peptide antigenic sites have been revealed in rat pancreatic B cells by applying immunohistochemical and cytochemical techniques. Fluorescein and rhodamine stains at the light-microscope level have detected both antigens in the same B cells. With the protein A-gold technique, labeling for both antigens was found in the cisternae of the rough endoplasmic reticulum, in those of the transitional elements, in all the cisternae of the Golgi apparatus except in the trans-most one, in the smooth but not in the coated vesicles, in the immature and mature secretory granules, and in some lysosomal (multigranular) structures. The fixation procedure used yielded excellent ultrastructural preservation which allowed for high resolution. The various control experiments demonstrated the high specificity of the results. Quantitative evaluations confirmed the qualitative observations in that they documented the specificity of the label and revealed the presence of an increasing gradient for both antigenic sites along the endoplasmic reticulum-Golgi-granule secretory pathway. The quantification also demonstrated various sites in which an increased labeling occurs: the rough endoplasmic reticulum, the smooth vesicles, the trans-cisternae of the Golgi apparatus, and the immature and the mature secretory granules. The Golgi apparatus was composed of three different subcompartments distinguished by their concentration of label. These include the cisternae on the cis-side, those on the trans-side, and the trans-most rigid cisternae. Since insulin and C-peptide form the proinsulin chain, their antigenic sites were found in the same locations along the secretory pathway; differences in location appeared only in the secretory granules, where insulin was concentrated in the core, while C-peptide was found in both the core and the halo of the granules. Furthermore, in the mature secretory granules displaying a crystalline core, insulin was restricted to the core, while C-peptide was confined to the halo. These results are in accord with the biochemical data, which indicate that simultaneous localization of both antigenic sites in compartments upstream to the immature secretory granules reflects their presence in the form of proinsulin. However, upon dissociation of proinsulin into insulin and C-peptide, both antigenic sites are segregated in different locations. The peptides appear to share parallel pathways and a fate which includes secretion through exocytosis or degradation by the lysosomal system.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively. The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyelocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative. Our observations highly suggest that small Golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.     

DIFFERENTIATION OF MONOCYTES : Origin, Nature, and Fate of Their Azurophil Granules

The origin, content, and fate of azurophil granules of blood monocytes were investigated in several species (rabbit, guinea pig, human) by electron microscopy and cytochemistry. The life cycle of monocytes consists of maturation in bone marrow, transit in blood, and migration into tissues where they function as macrophages. Cells were examined from all three phases. It was found that: azurophil granules originate in the Golgi complex of the developing monocyte of bone marrow and blood, and ultimately fuse with phagosomes during phagocytosis upon arrival of monocytes in the tissues. They contain lysosomal enzymes in all species studied and peroxidase in the guinea pig and human. These enzymes are produced by the same pathway as other secretory products (i.e., they are segregated in the rough ER and packaged into granules in the Golgi complex). The findings demonstrate that the azurophil granules of monocytes are primary lysosomes or storage granules comparable to the azurophils of polymorphonuclear leukocytes and the specific granules of eosinophils. Macrophages from peritoneal exudates (72–96 hr after endotoxin injection) contain large quantities of lysosomal enzymes throughout the secretory apparatus (rough ER and Golgi complex), in digestive vacuoles, and in numerous coated vesicles; however, they lack forming or mature azurophil granules. Hence it appears that the monocyte produces two types of primary lysosomes during different phases of its life cycle—azurophil granules made by developing monocytes in bone marrow or blood, and coated vesicles made by macrophages in tissues and body cavities.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Summary Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively.The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyolocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative.Our observations highly suggest that small golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.In honour of Prof. P. van Duijn     

Keratan sulfate glycosaminoglycans in murine eosinophil-specific granules.

We examined the presence of sialyl glycoconjugates in specific granules from murine bone marrow eosinophils. Lectin cytochemistry using Maackia amurensis lectin II (MAL II) specific for sialyl alpha-2,3 galactose residues demonstrated positive labeling in both immature and mature specific granules. Pretreatment with Clostridium neuraminidase or keratanase II eliminated the positive labeling of MAL II in the specific granules. High iron diamine-thiocarbohydrazide-silver proteinate physical development (HID-TCH-SP-PD) staining, which is specific for sulfated glycoconjugates, also positively labeled immature specific granules lacking crystalloids but not mature granules with crystalloids. Pretreatment with a combination of chondroitinase ABC and keratanase, or a combination of chondroitinase ABC and keratanase II, eliminated the positive labeling obtained with HID-TCH-SP-PD. These results indicate that the sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan, which appears to be of the corneal type in view of its sensitivity to keratanase and keratanase II. (J Histochem Cytochem 47:481-488, 1999)     

CYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE ACTIVITY IN GRANULE FRACTIONS FROM RABBIT POLYMORPHONUCLEAR LEUKOCYTES

When rabbit peritoneal exudates (97% polymorphonuclear [PMN] leukocytes, 2% mononuclear cells) were fractionated by zonal sedimentation or isopycnic centrifugation, four fractions (A, B, C, and D) were obtained, as reported earlier. "A" consisted largely of PMN azurophil granules, "B" of PMN specific granules, and "D" of membranous elements. The source of the more heterogeneous "C" fraction (containing acid hydrolases) was uncertain. To gain further information on the nature of this fraction, cytochemical tests for acid phosphatase (AcPase) were carried out on the starting cells and on the fractions. In intact PMN, lead phosphate reaction product was found in Golgi complexes, perinuclear cisternae, and some azurophil granules (immature forms or disrupted mature forms) of a few cells. The specifics and the intact azurophils were not reactive. Reaction product was also found within Golgi cisternae, secondary lysosomes, and some of the azurophil granules of mononuclear cells. Observations on the A and B fractions confirmed those in situ regarding the localization of reaction product in disrupted PMN azurophils, its absence from specifics, and the latency of the enzyme activity in intact azurophils. In the C fraction, AcPase was found in three structures (a) Golgi cisternae, (b) dense bodies, and (c) small pleomorphic granules Comparison with the starting cells indicates that the Golgi complexes are probably derived from both PMN leukocytes and mononuclear cells, whereas the remaining elements resemble (in size, shape, and density) secondary lysosomes and azurophil granules of mononuclear cells. The results indicate that the bulk of the cytochemically detectable AcPase present in the C fraction is derived from mononuclear cells, rather than from PMN leukocytes     

Histochemical analysis of mucosubstances and cytochemical studies of the Golgi region and the secretory granules of the normal gerbil gallbladder principal cells.

The mucosubstances of the gallbladder epithelium of the Mongolian gerbil have been analyzed histochemically. The results indicated that they are composed of carboxylated and sulfated glycoproteins. A closer study of the principal cell structure with ultrastructural and cytochemical technique localized the glycoproteins to the glycocalyx, lysosomes, Golgi complexes, immature and mature secretory granules and basement membrane. Acid phosphatase activity was found in the lysosomes, immature granules and the irregular cisternae of the internal region of the Golgi apparatus. It is suggested that the acid-phosphatase-containing internal Golgi membranes may correspond to the GERL (Golgi-endoplasmic reticulumlysosomes).     

ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES : Two Types Derived from Opposite Faces of the Golgi Complex in Developing Granulocytes

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO₄. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (~800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (~500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.     

Defensin-rich granules of human neutrophils: characterization of secretory properties

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.     

高尔基体对小鼠卵母细胞体外发育进程的影响

目的初步探讨高尔基体在小鼠卵母细胞体外发育进程中的作用。方法布雷菲德菌素A（Brefeldin A,BFA）处理小鼠未成熟,成熟卵母细胞,利用特异性标记物阻COP标记高尔基体。激光扫描共聚焦显微镜观察BFA处理对高尔基体产生的影响;同时。观察并比较不同处理组小鼠未成熟／成熟卵母细胞的体外成熟率、孤雌激活率、体外受精率及2-细胞率。结果GV期卵母细胞经BFA处理后,高尔基体的形态和分布发生明显改变。其体外成熟率（2．5％）与对照组（70．4％）比较统计学差异显著（P〈0．001）;洗掉BFA后,其体外成熟率（67．2％）与对照组无统计学差异（P〉0．05）。另外,成熟卵母细胞经BFA处理后。其体外受精率及2．细胞率均与对照组差异无统计学意义（P〉0．05）。结论小鼠卵母细胞体外成熟的正常进行需要高尔基体主导的膜运输。而体外受精和受精卵卵裂过程中不需要功能性的高尔基体。     

Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells

We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.     

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.     

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicer's hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiéry's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.     

Stimuli which provoke secretion of azurophil enzymes from human neutrophils induce increments in adenosine cyclic 3'-5'-monophosphate

1. Stimuli for human neutrophils were divided into two classes on the basis of their ability to induce degranulation: complete secretagogues provoked release of both azurophil and specific granules, while incomplete secretagogues only induced release of specific granules. 2. Complete secretagogues, which possessed the ability to induce secretion of azurophil granules, also induced transient increments in total cellular cyclic AMP levels: incomplete secretagogues did not. 3. Complete secretagogues, unlike the incomplete variety, also induced further increments of cyclic AMP in prostaglandin E1-pretreated neutrophils. 4. Inhibition of lysosomal enzyme release by prostaglandin E1 was closely correlated with elevated levels of cyclic AMP induced by the prostaglandin alone, than with the much higher transient increment in cyclic AMP produced by stimulation of prostaglandin E1-treated cells. 5. Our results describe the first biochemical difference between neutrophil responses associated with secretion of azurophil granules, as opposed to specific granules: transient increments in cyclic AMP.     

Heterogeneous distribution of phospholipids in membranes along the secretory pathway in pancreatic B-cells

The membrane content in phospholipids along the secretory pathway in rat pancreatic B-cells was studied in situ by high-resolution cytochemistry, applying the recently introduced phospholipase A2-gold technique. The gold particles were mostly associated with cell membranes, and the various types of membranes were labeled to a different extent. Quantitation of the labeling over these membranes revealed a heterogeneous distribution of the labeling across the secretory pathway. This heretogeneity occurred mainly as a progressive, decreasing gradient in the first half of this pathway, between the rough endoplasmic reticulum and the mi-cisternae of the Golgi apparatus. The labeling density remained at a lower level in the trans-most Golgi cisternae and immature secretory granule membranes, to increase in the mature secretory granule membrane, where it reached the value found in the plasma membrane. These results provide evidence that the functional heterogeneity existing across the membrane forming the secretory pathway is parallelled by substantial changes in their phospholipid content.     

Unmasking and redistribution of lysosomal sulfated glycoconjugates in phagocytic polymorphonuclear leukocytes

Rabbit heterophil and human neutrophil primary granules contain sulfated glycosaminoglycans (GAGs) and acid phosphatase, which can be readily stained in immature but not mature lysosomes. To determine whether this loss of staining represents masking of reactive components or removal of these components, we examined rabbit heterophils to see if high-iron diamine (HID)-reactive sulfate and acid phosphatase staining reappears in phagocytic vacuoles. Rabbit heterophils, obtained by peritoneal lavage, were incubated in vitro with latex beads or Pseudomonas aeruginosa for 15-60 min. Pre-embedment HID staining was enhanced in thin sections of unosmicated specimens with thiocarbohydrazide and silver proteinate (TCH-SP). Phagocytosis of latex beads or bacteria was progressively more prominent with time. Primary granules that were degranulated or in the process of degranulating into phagocytic vacuoles demonstrated intense sulfate staining with large (13 +/- 7 nm) HID-TCH-SP stain deposits. Smaller (6 +/- 1 nm) HID-TCH-SP stain deposits were present in tertiary granules, which were less frequently observed degranulating into phagosomes. Acid phosphatase staining was most intense during early phagolysosome formation. HID-TCH-SP staining was also observed in extracellular degranulated lysosomal matrices and on the surface of many peritoneal heterophils. These results indicate that loss of sulfate staining in mature heterophil granules is the result of masking by intragranular substances rather than of removal, and that these components may be unmasked during phagocytosis and/or redistributed to the cell surface after exocytosis.     

The processing and intracellular transport of myeloperoxidase. Modulation by lysosomotropic agents and monensin

Myeloperoxidase, stored in azurophil granules of neutrophils, is synthesized in promyelocytes as a larger molecular weight precursor, which is processed to yield a transient Mr 82 000 intermediate and mature polypeptides with molecular weights of 62 000 and 12 000. We have tried to define subcellular sites for processing using metabolic labelling of the promyelocytic leukemia cell line HL-60 in combination with subcellular fractionation on a Percoll gradient. A reasonable separation was achieved between azurophil granules, Golgi elements and endoplasmic reticulum. The finding of almost exclusively fully processed myeloperoxidase in granules and a mixture of unprocessed and processed polypeptide in fractions enriched in Golgi elements suggests that processing occurred mainly in pregranular structures. Monensin, which exchanges protons for Na+, and the base chloroquine blocked processing probably by inhibition of transport through the Golgi apparatus. However, the lysosomotropic NH4+ cation did not inhibit processing or transport indicating that processing is not necessarily influenced by pH-dependent mechanisms. Results from digestion with endoglycosidase H, incubation with tunicamycin and metabolic labelling with [3H]mannose indicated that myeloperoxidase contained high mannose oligosaccharide side chains. Also [32P]phosphate incorporated into Mr 90 000 and Mr 62 000 myeloperoxidase was susceptible to endoglycosidase H indicating that oligosaccharide side chains are modified by phosphorylation as in lysosomal enzymes. Thus, even if myeloperoxidase contained mannose 6-phosphate residues, these may not necessarily be involved in directing transport to the azurophil granules.     

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.