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H J Geuze

Publications and source records attributed to H J Geuze.

At least 127 records · Page 7Linked to original sources
PubMed1988

The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum.

We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome-associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1-anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.

Biological Transport↗
PubMed1988

Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes.

The distribution of CuZn superoxide dismutase (SOD) molecules in subcellular organelles in rat liver hepatocytes was studied using integrated biochemical, stereological, and quantitative immunocytochemical techniques. A known concentration of purified CuZn SOD in 10% gelatin was embedded alongside the liver tissue for the calculation of CuZn SOD concentrations in hepatocyte organelles and total CuZn SOD in the rat liver. Most of the CuZn SOD was located in the cytoplasmic matrix (73.1%) and in the nucleus (11.9%) with concentrations of 1.36 and 0.71 mg/cm3, respectively. Lysosomes contained the highest concentration (5.81 mg/cm3). Only low concentrations were measured in mitochondria (0.21 mg/cm3). Membrane-bound spaces of rough endoplasmic reticulum (ER), smooth ER, and the Golgi system did not contain significant concentrations of the enzyme. By adding up the concentrations in all subcellular compartments, a total liver content of CuZn SOD was established from the immunocytochemical measurements (0.386 +/- 0.028 mg/gm liver) that agreed closely with those obtained by biochemical assays (0.380 +/- 0.058 mg/gm liver). The average distances between two CuZn SOD molecules can be calculated from enzyme concentrations. It was determined that CuZn SOD molecules in the cytoplasmic matrix and nucleus were 34 and 42 nm apart, respectively. In peroxisomes and mitochondria, average intermolecular distance increased to approximately 60 nm and increased to 136 nm in smooth ER. CuZn SOD is a relatively abundant protein in the cytosol of hepatocytes and its distribution overlaps with major sites of O2- production. The efficiency of protection CuZn SOD can provide to cytosolic proteins from attacks by superoxide anion depends on the rate of O2- production, distribution of CuZn SOD relative to cytosolic proteins, and the relative reaction rates between O2- with both cytosolic proteins and CuZn SOD. Future studies of these substrate-enzyme relationships in vivo can lead to a greater understanding of how cells handle oxidant stress.

Animals↗
PubMed1988

Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles.

The intracellular distributions of the cation-independent mannose 6-phosphate receptor (MPR) and a 120-kD lysosomal membrane glycoprotein (lgp120) were studied in rat hepatoma cells. Using quantitative immunogold cytochemistry we found 10% of the cell's MPR located at the cell surface. In contrast, lgp120 was not detectable at the plasma membrane. Intracellularly, MPR mainly occurred in the trans-Golgi reticulum (TGR) and endosomes. lgp120, on the other hand, was confined to endosomes and lysosomes. MPR was present in both endosomal tubules and vacuoles, whereas lgp120 was confined to the endosomal vacuoles. In cells incubated for 5-60 min with the endocytic tracer cationized ferritin, four categories of endocytic vacuoles could be discerned, i.e., vacuoles designated MPR+/lgp120-, MPR+/lgp120+, MPR-/lgp120+, and vacuoles nonimmunolabeled for MPR and lgp120. Tracer first reached MPR+/lgp120-, then MPR+/lgp120+, and finally MPR-/lgp120+ vacuoles, which are assumed to represent lysosomes. To study the kinetics of appearance of endocytic tracers in MPR-and/or lgp120-containing pools in greater detail, cells were allowed to endocytose horse-radish peroxidase (HRP) for 5-90 min. The reduction in detectability of MPR and lgp120 antigenicity on Western blots, due to treatment of cell homogenates with 3'3-diaminobenzidine, was followed in time. We found that HRP reached the entire accessible pool of MPR almost immediately after internalization of the tracer, while prolonged periods of time were required for HRP to maximally access lgp120. The combined data suggest that MPR+/lgp120+ vacuoles are endocytic vacuoles, intermediate between MPR+/lgp120-endosomes and MPR-/lgp120+ lysosomes, and represent the site where MPR is sorted from lgp120 destined for lysosomes. We propose that MPR is sorted from lgp120 by selective lateral distribution of the receptor into the tubules of this compartment, resulting in the retention of lgp120 in the vacuoles and the net transport of lgp120 to lysosomes.

Animals↗
PubMed1988

Immunogold determination of amylase concentrations in pancreatic subcellular compartments.

We used the immunogold method on ultrathin cryosections to measure intracellular amylase (Am) concentrations in subcellular compartments of rat exocrine pancreatic cells. Previously, the quantitation procedure was characterized in a model system consisting of Am dispersed at known concentration in a matrix of gelatin. Variations in labeling efficiency, due to differences in matrix density, were equalized by embedding in 30% polyacrylamide (PAA). Here we applied these model conditions to rat pancreas and established intracellular Am concentrations [Am]. Specimen blocks were composed of tissue and a reference layer of gelatin mixed with a known Am concentration ([Am]r), both fixed in glutaraldehyde. Cryosections of the PAA embedded blocks were immunogold labeled for Am. The labeling density was measured in the reference layer (LDr) and in structures in exocrine cells that were involved in Am synthesis and transport (LDs). In each of these structures the Am concentration ([Am]s) was calculated from: [Am]s = [Am]r. LDs/LDr In this way we measured average concentrations ranging from 63 mg/ml in rough endoplasmic reticulum to 261 mg/ml in secretory granules. Concentration of Am appeared to occur mainly in the most cis- and the most trans-Golgi cisternae. To check whether sterical hindrance was an inherent bias to the [Am] measurements in compartments that contained high concentrations of the enzyme, the labeling efficiency for Am in intact isolated secretory granules in gelatin and embedded in PAA, was compared with the efficiency when the granules were lysed and approximately 50 times diluted in gelatin before PAA embedment. It appeared that Am was detected with similar efficiency under both conditions. This demonstrated that sterical hindrance did not cause errors in the measurements of cellular Am concentrations.

Amylases↗
PubMed1988

The two mannose 6-phosphate receptors have almost identical subcellular distributions in U937 monocytes.

Using a semiquantitative immunogold technique on ultrathin cryosections, the in situ subcellular distributions of the cation-dependent, 46-kDa mannose 6-phosphate receptor (small MPR) and of the cation-independent, 270-kDa mannose 6-phosphate receptor (large MPR) were for the first time compared. U937 cells were chosen because of their relatively high content of both receptor species. Of each receptor, about 12% occurred at the cell surface, 2% in the Golgi stack, and about 25% in vacuoles resembling endosomal vacuoles. About half of both receptors was found in tubules, presumably belonging to endosomes and trans-Golgi reticulum. It was concluded that the distribution of the small and large MPR were roughly similar. The only exception was formed by electron-dense vesicles occurring in the trans-Golgi region and surrounding endosomes. Dense vesicles contained significantly less small MPR (7%) than large MPR (12%).

Cells, Cultured↗
PubMed1988

Immunogold localization of circumsporozoite protein of the malaria parasite Plasmodium falciparum during sporogony in Anopheles stephensi midguts.

The occurrence of the circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was monitored during sporogonic development in Anopheles stephensi mosquitoes. Using a monoclonal anti-CS protein antibody (3Sp2) and immunogold labeling on ultrathin cryosections it was found that CS protein is synthesized in immature oocysts from day 6 onwards when there are not yet signs of sporozoite formation. The CS protein is rapidly incorporated in the oocyst plasmalemma, which subsequently invaginates into the parasite. In the oocyst only the external sporozoite membrane contains CS protein. The inner pellicle membranes, rhoptries and micronemes do not react with monoclonal antibody (MoAb) 3Sp2.

Animals↗
PubMed1987

Immunogold cytochemistry of cytochromes P-450 in porcine adrenal cortex. Two enzymes (side-chain cleavage and 11 beta-hydroxylase) are co-localized in the same mitochondria.

In order to study the distribution of mitochondrial cytochromes P-450 in porcine adrenal glands, the glands of anesthetized pigs were fixed in situ. Polyclonal antibodies against two cytochromes P-450, i.e., C27 side-chain cleavage enzyme and 11 beta-hydroxylase, were used to study the distribution of these enzymes in cryosections of the adrenal cortex. Ultrathin cryosections were evaluated by both protein-A/gold/silver immunocytochemistry and immunoelectron microscopy using double labeling with protein-A/colloidal-gold. At light microscopy, the two cytochrome P-450 enzymes were found to be broadly distributed in both the fasciculata and glomerulosa zones of the adrenal cortex. Quantitative immunoelectron microscopy revealed that both enzymes were localized only in mitochondria, in which they were present on the inner aspects of the inner mitochondrial membrane. Both cytochromes P-450 were demonstrable in all of the mitochondria examined, and statistical evaluation of the ratios of the two enzymes present in individual mitochondria yielded a normal distribution curve. Since no evidence was found for the preferential localization of either enzyme in a special population of mitochondria, we conclude that all mitochondria of the adrenal cortex contain both enzymes. We discuss implications of these findings with respect to the regulation of steroidogenesis.

Adrenal Cortex↗
PubMed1987

Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells.

After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'-diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both ligands in the same compartment. At 37 degrees C, sorting of the two ligands occurred with a half-time of approximately 2 min, and was nearly completed within 10 min. The 125I-ASOR/HRP-induced shift of 131I-transferrin was completely dependent on the receptor-mediated uptake of 125I-ASOR/HRP in the same compartment. In the presence of a weak base (0.3 mM primaquine), the recycling of transferrin receptors was blocked. The cell surface transferrin receptor population was decreased within 6 min to 15% of its original size. DAB cytochemistry showed that sorting between endocytosed 131I-transferrin and 125I-ASOR/HRP was also blocked in the presence of primaquine. These results indicate that transferrin and asialoglycoprotein are taken up via the same compartments and that segregation of the transferrin-receptor complex and asialoglycoprotein occurs very efficiently soon after uptake.

Asialoglycoprotein Receptor↗
PubMed1987

Ligand- and weak base-induced redistribution of asialoglycoprotein receptors in hepatoma cells.

The receptor for asialoglycoproteins (ASGPR) was localized in human hepatoma Hep G2 cells by means of quantitative immunoelectron microscopy. Without ligand added to the culture medium, we found 34% of the total cellular receptors on the plasma membrane, 37% in compartment of uncoupling receptor and ligand (CURL), and 21% in a trans-Golgi reticulum (TGR) that was defined by the presence of albumin after immuno-double labeling. A small percent of the ASGPR was associated with coated pits, the Golgi stacks, and lysosomes. After incubation of the cells with saturating concentrations of the ligand asialo-orosomucoid (ASOR), the number of cell surface receptors decreased to 20% of total cellular receptors, whereas the receptor content of CURL increased by a corresponding amount to 50%. The ASGPR content of TGR remained constant. In contrast, after treatment of the cells with 300 microM of the weak base primaquine (PMQ), cell surface ASGPR had decreased dramatically to only 4% of total cellular receptors whereas label in the TGR had increased to 42%. ASGPR labeling of CURL increased only to 47%. The labeling of other organelles remained unchanged. This affect of PMQ was independent of the presence of additional ASOR. Implications for the intracellular pathway of the ASGPR are discussed.

Asialoglycoprotein Receptor↗
PubMed1987

Membranes of sorting organelles display lateral heterogeneity in receptor distribution.

This study describes the distribution of an intrinsic membrane protein, the asialoglycoprotein receptor (ASGP-R) in the trans-Golgi reticulum and compartment of uncoupling receptor and ligand (CURL) of rat liver cells. Using quantitative immunogold electron microscopy and membrane length measurements, we showed lateral nonhomogeneity of receptors in the membranes of trans-Golgi reticulum and CURL, in particular in the membranes of secretory vesicles (identified by their content of albumin and very low density lipoprotein particles) and of CURL vesicles (endosomes), including multivesicular bodies. The characteristic tubulovesicular morphology of both sorting organelles defines the transition of receptor-rich tubular membrane and the receptor-poor limiting membrane of the attached vesicles. There was a direct relationship between the size of the secretory and CURL vesicles and the density of ASGP-Rs in their membranes. Receptor density in the smallest vesicles was similar to that found in adjacent continuous tubules. The larger the vesicles, the less receptor was detectable in their membranes. We propose that the receptor molecules are excluded from the vesicle membranes by dynamic lateral redistribution. Nonrandom receptor distribution in the CURL vesicle membranes was present even at the multivesicular body stage. These observations strongly suggest the existence of barriers to ASGP-R diffusion at the junctions of tubules and vesicles. In addition, our observations suggest that ASGP-Rs are transported to the plasma membrane via a mechanism other than the normal secretory pathway.

Albumins↗
PubMed1987

Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections.

We used a model system to study whether measurements of absolute local antigen concentrations at the electron microscopic level are feasible by counting immunogold labeling density in ultra-thin sections. The model system consisted of a matrix of a variable concentration of gelatin, which was mixed with given concentrations of rat pancreas amylase and fixed according to various fixation protocols. With a relatively mild fixation, there was no clear proportionality between anti-amylase gold labeling and amylase concentration in ultra-thin cryosections. This was presumably due to uncontrolled loss of amylase from the sections. After stronger fixation with 2% glutaraldehyde for 4 hr, labeling density reflected the amylase concentration very well. We observed that matrix (gelatin) density influenced labeling density. A low gelatin concentration of 5% allowed penetration of immunoreagents into the cryosection, resulting in a high and variable labeling density. In gelatin concentrations of 10% and 20%, labeling density was lower but proportional to amylase concentration. To establish an equal (minimal) penetration of immunoreagents, we embedded model blocks with different matrix densities in polyacrylamide (PAA). In ultra-thin cryosections of these PAA-embedded blocks, anti-amylase labeling was proportional to amylase concentration even at a low (5%) gelatin concentration. Anti-amylase labeling in ultra-thin sections from Lowicryl K4M low temperature-embedded blocks was higher than in PAA sections, but the results were less consistent and depended to some extent on matrix density. These results, together with the earlier observation that acrylamide completely penetrates intracellular compartments (Slot JW, Geuze HJ: Biol Cell 44:325, 1982), demonstrate that it is possible to measure true intracellular concentrations of soluble proteins in situ using ultra-thin cryosections of PAA-embedded tissue.

Acrylic Resins↗
PubMed1987

On the effects of weak bases and monensin on sorting and processing of lysosomal enzymes in human cells.

The weak bases chloroquine, primaquine, NH4Cl and the ionophore monensin exert similar but not identical effects on sorting, transport and processing of cathepsin D in several human cell lines (fibroblasts, HepG2 cells, U937, monocytes). The drugs inhibit the segregation of newly synthesized cathepsin D from the secretory route. The kinetics of transport of nonsegregated cathepsin D precursor along the secretory route is retarded resulting in a delayed hypersecretion. Higher concentrations of the drugs can arrest the intracellular transport completely. The extent of inhibition of segregation varies among the different human cell types tested. Thus, in fibroblasts the secretion can be stimulated to exceed 80%, while in U937 cells the secretion cannot be enhanced above 50% although both cell types have the same basal rate of secretion (approximately 10% of the synthesized cathepsin D). We suggest that pH-independent sorting mechanisms contribute to the targeting of cathepsin D in U937 cells. Processing of the cathepsin D remaining in cells is characteristically changed depending on the drug. The proteolytic processing is strongly inhibited by chloroquine and is rather insensitive to monensin. Unlike the other drugs, monensin blocks the formation of complex oligosaccharides in cathepsin D and allows for extensive secretion solely of molecules that are sensitive to endo H.

Ammonium Chloride↗
PubMed1986

Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line.

The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.

5'-Nucleotidase↗
PubMed1986

Immunogold localization of procollagen III, fibronectin and heparan sulfate proteoglycan on ultrathin frozen sections of the normal rat liver.

In the present study, we have localized by immunocytochemistry at the LM and EM level, procollagen type III (PIIIP), fibronectin (FN) and heparan sulfate proteoglycan (HSPG). Intracellularly, PIIIP was observed in both parenchymal and endothelial cells. In parenchymal cells, PIIIP was found in Golgi derived vesicles. This observation suggests that PIIIP synthesis is a normal function of liver parenchymal cells. In endothelial cells, vesicles, which could not be identified, were seen to contain PIIIP. This result does not allow to conclude, whether sinusoidal endothelial cells secrete or take up PIIIP. Extracellularly, PIIIP was present around portal and central veins, in the space of Disse and between adjacent parenchymal cells. In the space of Disse, almost all interstitial collagen fibrils reacted with the anti PIIIP antibodies. This observation leads to the conclusion that most fibrils of the space of Disse contain type III in addition to type I collagen molecules. By immunofluorescence, FN was seen mainly along the sinusoids in discrete dots. By EM, FN was found to be present in diffuse material closely associated with the sinusoidal membrane of the parenchymal cells and in strands connecting adjacent parenchymal cells, parenchymal and endothelial cells or parenchymal cells and collagen fibrils. FN was also present in vascular and ductular basal laminae. Strong HSPG reaction was observed around bile ducts. Moderate reaction was seen around blood vessels and in the space of Disse. In the latter location, the ultrastructural distribution of HSPG resembles that of FN, i.e. HSPG is present in diffuse material and in strands.

Animals↗
PubMed1986

Presence of asialoglycoprotein receptors in the Golgi complex in the absence of protein synthesis.

In rat hepatocytes the Golgi complex contains a considerable amount of receptors for asialoglycoproteins (ASGP-R). To establish whether the presence of ASGP-R in the Golgi complex originate from de novo synthesis isolated rat hepatocytes were incubated with 100 micrograms/ml cycloheximide to stop protein synthesis. Provided that protein synthesis was completely inhibited by cycloheximide, uptake and degradation of ligand (asialo-orosomucoid) were unaffected. Also intracellular transport of newly synthesized proteins, as determined by monitoring biosynthesis and intracellular transport of albumin and ASGP-R, was not affected. After culturing the cells for 3.5 h in the presence of cycloheximide, no more albumin could be detected in the Golgi complex with immunofluorescence microscopy. However, immunocytochemical assessment showed that the ASGP-R was still in the Golgi complex. These results suggest that the Golgi complex contains a pool of ASGP-R which is independent of neosynthesis for several hours.

Albumins↗
PubMed1986

Epitopes of apolipoprotein B-100 and B-48 in both liver and intestine. Expression and evidence for local synthesis in recessive abetalipoproteinemia.

The presence of apolipoprotein (apo) B in liver and intestine from a patient with abetalipoproteinemia was evaluated by immunohistochemistry with a polyclonal and six monoclonal antibodies to different apo B-48 and B-100 epitopes. In normal liver, apo B was present inside and outside hepatocytes. The patients liver exhibited staining in the cytoplasm with the polyclonal and three monoclonal antibodies. By immunoelectron-microscopy, apo B was found to be present in the smooth endoplasmatic reticulum and the Golgi complex. Normal intestinal epithelium was labeled with polyclonal and all monoclonal antibodies, including those specific for apo B-100. The patients epithelium stained with polyclonal and six monoclonals, and apo B was present in the Golgi complex. Thus, normal intestinal mucosa expressed apo B-48 and B-100 epitopes, which indicates apo B-100 synthesis in the gut. The synthesis of the apo B molecule in the patient seems to be retained in both liver and gut, which suggests a posttranslational defect.

Abetalipoproteinemia↗
PubMed1986

A quantitative immuno-electronmicroscopic study of amylase and chymotrypsinogen in peri- and tele-insular cells of the rat exocrine pancreas.

Malaisse-Lagae demonstrated in 1975 that peri-insular (PI) cells and tele-insular (TI) cells produce amylase (Am) and chymotrypsinogen (Ch) in a different ratio. These biochemical measurements are in contradiction with recent observations of Bendayan (1985), who found that the Am/Ch ratio measured with the protein A-gold technique applied to ultrathin Epon sections was the same in PI and TI cells. We have previously shown (Posthuma et al., 1984) that experimentally induced changes in Am and Ch content of rat pancreas are quantitatively reflected by immuno-gold labeling of zymogen granules in cryosections. Here we applied the same technique to compare the Am/Ch labeling density ratios in PI and TI pancreatic cells. To ascertain constancy of experimental conditions, we used ultrathin cryosections from tissue blocks consisting of TI and PI tissue elements. Consecutive sections of these blocks were alternatively immunolabeled for Am and Ch, using protein A-gold as marker. The density of gold particles over zymogen granules of both PI and TI cells was measured. It appeared that the Am/Ch labeling density ratio was significantly lower in PI than in TI cells. This difference resulted from a lower Am labeling as well as higher Ch labeling density over zymogen granules in PI cells.

Amylases↗