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Background & Aims Hepatocellular ballooning is a key finding in nonalcoholic steatohepatitis (NASH). It is conventionally defined by hemotoxylin and eosin (H&E) staining showing enlarged cells with rarefied cytoplasm and recently by changes in the cytoskeleton. Fat droplets are emerging as important organelles in cell metabolism. To address a possible relation between fat droplets and ballooning, we studied fat staining, H&E, and keratin 18 staining in human NASH. Methods Sequential staining and high resolution imaging were used to study freshly prepared cryo-sections from 10 patients with histologically confirmed steatohepatitis using oil red O for fat droplet identification, H&E to identify ballooning, and anti-K18 to confirm cytoskeletal changes. High resolution images were captured at each stage using the Aperio Scanscope. To provide ultrastructural correlation, glutaraldehyde-fixed specimens were studied using transmission electron microscopy (TEM) with serial sectioning for localization of ballooned cells by light microscopy and TEM in identical specimens. Results Serial staining consistently demonstrated that hepatocellular ballooning is associated with fat droplet accumulation evident by oil red O positivity and depletion of cytoplasmic keratin 18 with K-18 positive Mallory–Denk bodies (MDB). TEM confirmed the association between osmium stained fat droplets, MDB formation, and cellular enlargement and suggested droplet-associated dilation of the endoplasmic reticulum. Conclusions These results indicate a relationship between cellular ballooning, fat droplet accumulation, and cytoskeletal injury in NASH. We speculate that injury to multiple, organelles including fat droplets and endoplasmic reticulum, contribute to this characteristic finding.

OBJECTIVES: Troglitazone is a thiazolidinedione and peroxisome proliferator–activated receptor gamma (PPARγ) ligand used to treat diabetes mellitus type II. Because hyperinsulinemia may be a factor in nonalcoholic steatohepatitis (NASH), we postulated that troglitazone could have beneficial effects in this disorder. Our study was initiated before reports of idiosyncratic hepatitis induced by this agent and was completed before its recent withdrawal from the market. METHODS: We studied 10 female patients (age 44 ± 16) with histological NASH. All but two were obese (mean body mass index, BMI = 38 ± 6). One had type 2 diabetes, and three had well-compensated cirrhosis with NASH. Troglitazone was given at a dose of 400 mg/day for ≤6 months. Responders (defined as normal ALT at the end of treatment) were rebiopsied. Paired specimens were compared in blinded fashion. Mitochondria were quantitated using ultrathin electron microscopy. RESULTS: Seven of ten patients responded with normal ALT at the end of treatment. One of three nonresponders initially normalized ALT but returned to pretreatment level at 3 months. In this patient, therapy was stopped, and the ALT has remained at the baseline level with no other clinical or laboratory findings. In the responders, ALT fell from 87 ± 38 before to 39 ± 9 at the end of treatment (p = 0.01), and AST decreased from 77 ± 23 to 30 ± 8 (p = 0.002). Biopsy comparisons before and after therapy showed persistent steatohepatitis in all cases, although four of seven showed a one-point improvement in the necroinflammatory grade. Electron microscopy revealed elongation of the mitochondria after therapy. CONCLUSIONS: Normal ALT was seen in 70% of NASH patients at the end of treatment, but this biochemical response was associated with only mild histological improvement, and all follow-up biopsies had evidence of NASH. Normalization of the liver enzymes in patients with NASH who are treated with thiazolidinediones should be viewed with reservation. Follow-up biopsy is essential to evaluate the efficacy of these agents, which, at the histological level, appears to be relatively modest.

Background —In patients with recurrent persistent atrial fibrillation (AF), vulnerability to AF persists indefinitely despite presumed completion of reverse electrical remodeling within days of return to normal sinus rhythm. Atrial electrical and anatomic remodeling and reverse remodeling were studied in a canine model of chronic AF. Methods and Results —Chronic AF was induced in 8 dogs by creating moderate mitral regurgitation and rapidly pacing the right atrium at 640 bpm for >8 weeks. Measurements performed at baseline, after establishment of chronic AF, and then at 4 hours and again at 7 to 14 days after cardioversion to sinus rhythm included atrial effective refractory periods, AF cycle lengths, left atrial dimensions, premature atrial contraction (PAC) frequency, and atrial vulnerability to atrial extrastimuli. After establishing chronic AF, atrial effective refractory period shortening, increases in spontaneous PAC frequency, increases in left atrial size with loss of contractility, and multiple ultrastructural abnormalities were demonstrated. Complete reverse electrical remodeling and decreases in PACs were observed after 7 to 14 days of sinus rhythm, but there was no resolution of anatomic and ultrastructural abnormalities. Occurrence of spontaneous AF paralleled PAC frequency, but vulnerability to AF induction persisted (75% immediately after conversion versus 63% at 4 hours and 50% at 7 to 14 days) despite reverse electrical remodeling. Conclusions —After conversion from chronic AF to sinus rhythm in this canine model, electrical remodeling occurs rapidly. However, gross and ultrastructural anatomic changes persist, as does vulnerability to induced AF. Vulnerability to AF initiation 7 to 14 days after cardioversion is more dependent on persisting structural abnormalities than on electrophysiological abnormalities.

Use of antibiotic-impregnated spacers is common in the two-stage approach to treatment of periprosthetic joint infection despite the lack of information regarding in vivo performance of these implants. Antibiotic elution levels likely often fall below the minimal inhibitory concentration need to inhibit bacterial growth, raising concern that the spacers themselves may provide a potential attachment site for biofilm formation. Advanced microscopy was used in this study to evaluate the surface characteristics of antibiotic-eluting spacers collected at the time of prosthesis reimplantation from 6 patients undergoing two-stage treatment for an infected total joint arthroplasty. Scanning electron microscopy and confocal scanning microscopy of the removed spacers revealed modest fibrous matrix formation and inflammatory cells with no biofilm or bacteria detected. This study supports the continued use of antibiotic spacers in the treatment of periprosthetic joint infection.

Ultrastructural examination of the hepatocyte in disease states has been an important research tool for many years [1]. Although eclipsed by techniques of molecular biology, electron microscopy (EM) remains a useful and insightful tool. EM can inform various aspects of fat metabolism in fatty liver disease, including alcoholic fatty liver, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Limitations of EM include small sample size, artifact introduced by variations in the technique of sample processing, laborintensive methods and a paucity of normal tissue controls in human studies [2]. However, refinements in technique and technology have evolved into reliable methodology, and EM offers an intriguing perspective on cellular physiology. In this article, we highlight key observations reported in human NASH, along with potential pathophysiologic correlations.

Isolation of glioblastoma stem cells requires incubation of tumor cells in a neural stem cell media. Neurospheres containing these glioblastoma stem cells are formed after approximately a five-day period. These cells can then be analyzed for the presence of stem cell markers. Immunofluorescence staining for these markers can serve as a valuable tool for analyzing the intact neurosphere directly in stem cell media. Here we present the use of a novel fixative (1,4-benzoquinone) for immunoflourescence staining of neurospheres.

Mitochondrial dysfunction is an important element in the pathogenesis of nonalcoholic steatohepatitis (NASH). Intramitochondrial crystals (IMCs) are a well-documented morphological abnormality seen on transmission electron microscopy in this disease. It has been suggested that IMCs consist of phospholipids, but their exact composition remain uncertain many years after their discovery. Micellar phase transitions of phospholipid bilayers is a well-known but little-studied phenomenon in living systems. Its presence in the mitochondria of NASH would offer significant insight into the disease with possible therapeutic implications. We postulated that intramitochondrial disturbances in NASH are sufficient to produce such transitions and that their detection in fresh biopsies would therefore be a dynamic process. To test this, we performed a blinded, prospective analysis of fresh liver biopsy samples immediately fixed under different conditions. Quantitative transmission electron microscopy morphometry, performed by systematically counting total mitochondria and IMCs within areas of uniform dimension, showed a stepwise decline in IMCs with cooler fixation temperature in each subject studied. Randomization testing (Monte Carlo resampling) confirmed that the detection of IMCs was strongly dependent on fixation temperature (P < 0.0001). Conclusion: These results indicate that the intramitochondrial crystals characteristic of NASH are highly dynamic and unstable structures. The findings offer the strongest support yet for their origin in micellar phase transitions. We speculate that such transitions result from microenvironmental changes within the mitochondria and carry therapeutic implications, especially in regard to dietary manipulations of mitochondrial lipid composition.

Enlarged Hepatocytes in NAFLD Examined with Osmium Fixation: Does Microsteatosis Underlie Cellular Ballooning in NASH?

Nonalcoholic fatty liver (NAFL) or nonalcoholic fatty liver disease (NAFLD) as it is often called, is increasingly recognized as a common and potentially severe condition often associated with obesity, type 2 diabetes, and hyperlipidemia. The spectrum of disorders which fall under the term ‘NAFL’ include simple steatosis, steatosis with mild inflammation, and steatosis with inflammation and varying degrees of fibrosis. Most investigators reserve the term ‘NASH’ (nonalcoholic steatohepatitis) for those patients who have some degree of fibrosis, usually associated with increased numbers of ballooned hepatocytes. The prognosis varies, but it is now evident that a substantial portion of patients, especially those with NASH, will progress to cirrhosis with all of the attendant complications, and some may ultimately develop hepatocellular cancer. Well-developed cirrhosis may lose its fat content and appear as ‘cryptogenic cirrhosis’. Lipid peroxidation of the excess oil in the liver appears to be the major pathogenic mechanism. The process appears to alter mitochondrial form and function. Initial treatment usually involves exercise and dietary modifications. Patients who fail this approach or who have more advanced conditions initially may be candidates for more aggressive measures, including drug therapy. Many agents have been reported in small series but all remain investigational.

Megamitochondria with crystalline inclusions (MMC) have been previously described in nonalcoholic fatty liver; however, their distribution within hepatic zones is unknown. We sought to determine this distribution from the core liver biopsy specimens of 31 patients: 8 males and 23 females, age range 21 to 72 years. Twenty-nine showed evidence of nonalcoholic steatohepatitis (NASH) on biopsy with steatosis, inflammation, varying degree of fibrosis, ballooned hepatocytes, and Mallory hyaline, and two patients had cryptogenic cirrhosis thought to represent “burned out” NASH. Identified by transmission electron microscopy, the abundance of MMC was compared between low-stage (fibrosis stages 1 and 2) and high-stage (fibrosis stages 3 and 4) groups and between zones with or without difference in fibrosis stage. Regardless of stage, the MMC were distributed equally in all zones and were abundant similarly in low- and high-stage groups. This abundance did not correlate with the degree of oxidative stress (4-hydroxynonenal staining) or with the abundance of ballooned hepatocytes. Consistent with age as a risk factor for more severe disease, the median age for the low-stage group was significantly lower than that of the high-stage group (P = .003). In conclusion, in NASH, the MMC seem to be distributed randomly among zones and without variation in abundance, regardless of the fibrosis stage. The exact function of these structures remains to be defined. In this study, their presence did not seem to correlate with the light microscopic injury pattern represented by ballooned hepatocytes or degree of oxidative stress defined by immunostaining for 4-hydroxynonenal. (HEPATOLOGY 2004;39:1423–1429.)

OBJECTIVE: Our purpose was to study the expression and localization of parathyroid hormone– related protein and expression of the parathyroid hormone/parathyroid hormone– related protein receptor in human umbilical cord. STUDY DESIGN: The expression and localization of parathyroid hormone– related protein and expression of the parathyroid hormone/parathyroid hormone– related protein receptor were studied in isolated tissues from the human umbilical cord by Northern analysis, reverse-transcriptase polymerase chain reaction, Southern gel analysis, and immunolocalization procedures at the light and electron microscopic levels. RESULTS: Parathyroid hormone– related protein was abundantly expressed in the umbilical cord. Immunohistochemical and immunocytochemical techniques confirmed hormone localization in the amnion epithelial layer and in vascular smooth muscle and endothelial cells in vessels from the umbilical cord and placental chorionic plate. Multiplex reverse-transcriptase polymerase chain reaction identified expression of receptor messenger ribonucleic acid in vessels of the umbilical cord; this finding was verified by means of Southern gel analysis of the products of the reverse-transcriptase polymerase chain reaction. CONCLUSION: A parathyroid hormone– related protein paracrine system appears to exist in human umbilical cord. We suggest that it may be involved in the control of fetal placental circulation. (A M J O BSTET G YNECOL 1994; 170:1018-26.)

The intralobular distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c (P-450) reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) in rat liver has been investigated by means of two quantitative immunohistochemical techniques: microdensitometric quantitation of unlabeled antibody peroxidase-antiperoxidase staining and microfluorometric analysis of indirect fluorescent antibody staining. Utilizing sheep antiserum elicited against NADPH-cytochrome c (P-450) reductase that had been isolated and purified to apparent homogeneity from rat liver microsomes, the reductase was detected within hepatocytes throughout the liver. However, differences in the intensity of staining of hepatocytes within different regions of the liver lobule were readily apparent after completion of both immunohistochemical staining procedures. These visual findings were verified by microdensitometric and microfluorometric analyses of immunohistochemical staining, both of which revealed that approximately the same degree of staining for NADPH-cytochrome c (P-450) reductase was produced within the centrilobular and midzonal regions of the liver lobule, whereas periportal hepatocytes were stained with significantly less intensity. These results demonstrate that the application of either microdensitometry in conjunction with unlabeled antibody peroxidase-antiperoxidase staining or microfluorometry after indirect fluorescent antibody staining can be used to quantitatively determine the intratissue distributions of antigens.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.

Surgical excision of one of the compound eyes from juvenile crayfish leads to the regeneration of a heteromorphic antennule in 30% of the cases. Most of the heteromorphic antennules generated this way are bifurcate appendages possessing morphologically distinct medial and lateral branches. These structures are identical to the internal and external flagella of the normal antennules, and the homolog of the external flagellum bears aesthetascs supplied by olfactory sensory neurons. Autoradiographic analysis of the brain following exposure of heteromorphic antennules to tritiated leucine indicates that the supernumerary sensory axons transport the labeled amino acid into their central terminals at appropriate target locations within the ipsilateral olfactory lobe. The data suggest that olfactory input from heteromorphic antennules is incorporated into the organized central projection of olfactory afferents from the normal antennule.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochromes P -450 in adrenocortical mitochondria, has been localized immunohistochemically at the light microscopic level in rat adrenals by employing rabbit antiserum to bovine adrenal ferredoxin in both an unlabeled antibody peroxidase-antiperoxidase method and an indirect fluorescent antibody method. When sections of rat adrenals were exposed to the adrenal ferredoxin antiserum in both procedures, positive staining for adrenal ferredoxin was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. Marked differences in the intensity of staining, however, where observed among the three cortical zones: the most intense staining being found in the zona fasciculata, less in the zona reticularis, and least in the zona glomerulosa. Furthermore, differences in staining intensity were also observed among cells within both the zona fasciculata and the zona reticularis. In agreement with these immunohistochemical observations, determinations of adrenal ferredoxin contents by electron paramagnetic resonance (EPR) spectrometry in homogenates prepared from capsular and decapsulated rat adrenals revealed that the concentration of adrenal ferredoxin in the zona glomerulosa was lower than that in the zona fasciculata-reticularis. Similar results were obtained when the contents of cytochrome P -450 were determined in capsular adn decapsulated rat adrenal homogenates. These observations indicate that adrenal ferrodoxin and cytochrome P -450 are not distributed uniformly throughout the rat adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P -450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum procued against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase techniques and an indirect fluorecent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.

Antibodies produced against two forms of cytochrome P-450, PB-B and MC-B, which were purified to apparent homogeneity from hepatic microsomes of rats pretreated with phenobarbital and 3-methylcholanthrene, respectively, have been employed to localize these hemoproteins immunohistochemically at the light microscopic level in the livers of untreated rats. Using these antibodies in an unlabeled antibody peroxidase-antiperoxidase technique, immunohistochemical staining for the cytochromes P-450 was detected in parenchymal cells throughout the liver lobule. The patterns of immunohistochemical staining intensity observed with the two antibodies, however, were quite different. Exposure of liver sections to the antibody to cytochrome P-450 PB-B resulted in intense immunostaining within the centrilobular regions but produced staining of considerably weaker intensity in the peripheral regions of the lobule. In contrast to these observations, the antibody to cytochrome P-450 MC-B yielded a more uniform pattern of immunohistochemical staining, with the intensity of staining being only slightly greater in the centrilobular regions. The results of this immunohistochemical study thus demonstrate that different patterns of distribution exist for different forms of cytochrome P-450 within the liver lobule and that the greatest concentration of cytochrome P-450 occurs within the centrilobular regions of the liver.

NADPH-cytochrome c reductase (NADPH-cytochrome reductase, EC 1.6.2.4), the flavoprotein which is responsible for the NADPH-dependent reduction of cytochromes P-450 in hepatic microsomes, has been localized immunohistochemically at the light microscopic level in rat liver. Localization was achieved through the use of sheep antiserum to rat hepatic microsomal NADPH-cytochrome c reductase in an unlabeled antibody peroxidase-antiperoxidase technique. Parenchymal cells throughout the liver lobule were found to be stained positively for NADPH-cytochrome c reductase, although the intensity of immunostaining was slightly greater in the centrilobular regions. Immunostaining for NADPH-cytochrome c reductase was not detected in Kupffer cells, connective tissue cells, or in cells of the hepatic vasculature.

The superior cervical ganglion (SCG) of the guinea pig has been investigated by a multidisciplinary approach. Dopamine (50 μM) produced no increase in cyclic AMP levels above control values of 27.9 pmole/mg protein, but 50 μM isoproterenol produced cyclic AMP levels of 210 pmole/mg protein, indicating the existence of a β-adrenergic receptor-adenylate cyclase complex. The SIF cells were studied by fluorescence histochemistry, which indicated that two morphological types were present. A few Type I cells of the guinea pig SCG were solitary, but most were present in clusters containing many Type II cells. Immunohistochemical localization of antibodies to dopamine-β-hydroxylase (DBH) andphenylethanolamine-N-methyl-transferase (PNMT) demonstrated that both types of SIF cell localize antibodies to DBH but not PNMT, providing strong evidence that norepinephrine is the neurotransmitter for all the SIF cells of the guinea pig SCG. Determination of the ratio of norepinephrine to dopamine confirmed that no other dopamine pools exist in the guinea pig SCG.

Employing immunohistochemical techniques, the distributions of NADPH-cytochrome c (P–450) reductase and of two forms of cytochrome P–450, PB–B and MC–B, were examined in the livers of untreated rats and of rats pretreated for four days with phenobarbital, 3-methylcholanthrene, or pregnenolone-16α-carboni- trile. Fluorescent and peroxidase-antiperoxidase immunohistochemical staining revealed that each enzyme exhibits a characteristic pattern of intralobular distribution which is affected in characteristic manners by different xenobiotics.

Electron microscopic localization of dopamine-β-hydroxylase (DBH) was carried out in bovine adrenal medullary chromaffin granule fractions and in intact bovine adrenal medulla by means of a peroxidase-antiperoxidase unlabelled antibody technique according to Sternberger , Hardy , Cuculis and Meyer (1970). Adrenal medullary chromaffin granule fractions showed retention of DBH despite successive fractionation, fixation and prolonged washing procedures, indicating that a portion of the DBH in the chromaffin granule is quite tightly bound. Under these circumstances the electron-opaque granular matrix was absent, implying loss of the more soluble constituents of the chromaffin granules, including matrix-associated DBH. Intact bovine adrenal medulla which was fixed, sectioned and subjected to immunocytochemical localization of DBH showed the enzyme to be present throughout the matrix of the chromaffin granule and demonstrated that, under these less vigorous conditions, the electron-opaque granule matrix was not removed. It would therefore appear to be feasible to differentiate directly between the “soluble” and “bound” forms of DBH by immunocytochemical procedures and to apply this technique to elucidate the process of exocytosis in adrenal medulla and adrenergic nerve terminals.

An antibody produced against epoxide hydratase (EC 4.2.1.63) which had been purified to apparent homogeneity from hepatic microsomes of phenobarbital pretreated rats was employed in an unlabeled antibody peroxidase-antiperoxidase technique to localize the enzyme at the light microscopic level in the livers of untreated rats. Immunohistochemical staining for epoxide hydratase was detected in parenchymal cells throughout the liver lobule. Cells within the centrilobular regions, however, were observed to be stained more intensely than were those within the midzonal and periportal regions of the lobule. The results of this immunohistochemical study thus demonstrate that epoxide hydratase does not exhibit a uniform pattern of distribution within the liver lobule in untreated rats.

Prostaglandin E (PGE) has been localized via the unlabeled antibody technique in freeze-dried and ethanol-fixed cryostat sections. Discrete perivascular and stromal localization was present in the uterus prepared by the method presented, but not in classically fixed specimens. Absorption of the anti-PGE by addition of free PGE was ineffective; whereas, removal of PGE-reactive antibodies from the anti-serum was effectively accomplished with an Affigel-101-PGE immunoadsorbant column.

An indirect immunocytochemical technique (Nakane, 1970) was employed to localize the soluble proteins purified from lysates of catecholamine (CA)-containing vesicles of the bovine adrenal medulla. Antiserum to the proteins, produced in rabbits, was used for incubation of sections of bovine adrenal tissue prepared by fixation in glutaraldehyde and embedding in serum albumin (McLean & Singer, 1970). The site of the antigen-antibody complex was visualized by incubating the sections with anti-rabbit γ-globulin (goat) conjugated to peroxidase, followed by the deposition of electronopaque reaction product generated by the enzyme. The reaction product, also visible at the level of the light microscope, appeared to have a distribution similar to that of the CA-storage vesicles. Electron microscopic examination revealed that nearly all the reaction product was deposited over the electron-opaque core of the vesicles. The intravesicular localization is consistent with the proposal that these proteins exist primarily in the CA-containing granular vesicle and may function to stabilize the CA-storage complex.