Light microscopic immunocytochemical demonstration of peroxisomal enzymes in epon sections

A procedure is described for light microscopic immunocytochemical localization of catalase and three enzymes of peroxisomal lipid beta-oxidation: acyl-CoA oxidase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase in semi-thin sections of rat liver processed for routine electron microscopy. Satisfactory immunostaining required the removal of the epoxy resin with sodium ethoxide, controlled digestion of deplasticized sections with proteases and, in case of osmiumfixed tissue, bleaching with oxidants. Resin removal was essential for successful immunostaining, and protease treatment enhanced markedly the intensity of the reaction. This study shows that tissues processed for conventional ultrastructural studies can be used for postembedding immunocytochemical demonstration of various peroxisomal enzymes.     

Detection of peroxisomes in human liver and kidney fixed with formalin and embedded in paraffin: the use of catalase and lipid beta-oxidation enzymes as immunocytochemical markers

Summary We describe the immunocytochemical localization of four peroxisomal enzymes by light microscopy in human liver and kidney processed routinely by formalin fixation and paraffin embedding. Monospecific antisera against catalase and three enzymes of peroxisomal lipid -oxidation (acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase) were used in conjunction with either the indirect immunoperoxidase method or the protein A—gold technique followed by silver intensification. The sections of formalin-fixed paraffin-embedded tissue had to be deparaffinized and subjected to controlled proteolysis in order to obtain satisfactory immunostaining. Under the conditions employed, peroxisomes were distinctly visualized in liver parenchymal cells with no reaction in bile duct epithelial or sinusoidal lining cells. In the kidney, peroxisomes were confined to the proximal tubular epithelial cells with negative staining of glomeruli, distal tubules and collecting ducts. A positive immunocytochemical reaction was obtained even in paraffin blocks stored for several years. The method offers a simple approach for detection of peroxisomes and evaluation of their various enzyme proteins in material processed routinely in histopathology laboratories and should prove useful in the investigation of the role of peroxisomes in human pathology for both prospective and retrospective studies.     

Immunocytochemical demonstration of peroxisomal enzymes in human kidney biopsies

Peroxisomes are particularly abundant in the proximal tubules of the mammalian kidney. We describe the immunocytochemical localization of catalase and three peroxisomal lipid beta-oxidation enzymes: acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase, in human renal biopsies fixed with glutaraldehyde and embedded in Epon. For light microscopy of semithin sections, satisfactory immunostaining required removal of the resin and controlled proteolytic digestion followed by the indirect immunoperoxidase technique. Brief etching of ultrathin sections with alkoxide followed by the protein A-gold method were used for electron microscopic localization of the enzymes. The immunoreactive peroxisomes were distinctly visualized in proximal tubular epithelial cells with no staining of any other cell organelles. The results establish the presence of catalase and of peroxisomal lipid beta-oxidation system proteins in human kidney. The immunocytochemical procedure described herein provides a simple approach for the investigation of peroxisomal structure and function in human renal biopsies processed for ultrastructural studies.     

Immunocytochemical detection of peroxisomal β-oxidation enzymes in cryostat and paraffin sections of human post mortem liver

Summary The immunocytochemical visualization of the peroxisomal -oxidation enzymes was investigated in three human post mortem liver samples. Acyl-CoA oxidase, bifunctional protein and 3-oxoacyl-CoA thiolase remained immunocytochemically detectable 30, 55 and 72 h after death. Peroxisomes in the parenchymal cells were clearly visualized for light microscopy (paraffin and cryostat sections), using protein A-gold in combination with silver enhancement. In two samples catalase activity became very weak, but catalase antigenicity was well preserved. The findings prove the diagnOstic value of post mortem samples, even after extreme conditions of tissue conservation. The technique of immunocytochemical staining for the peroxisomal -oxidation enzymes on unmounted cryostat sections has not been reported previously. This method allows a quick diagnOsis of biopsies from patients suspected of peroxisomal disorders.     

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35–1.0 m) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 m. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.     

Quantitative immunocytochemical studies on differential induction of serine

Summary Differential induction of serine: pyruvate aminotransferase (SPT) in rat liver parenchymal cells by administration of glucagon or di-(2-ethylhexyl)phthalate (DEHP) was studied using post-embedding immunocytochemical techniques and morphometric methods. Two groups of rats were fasted for 5 days and daily received peritoneal injection of glucagon (300 g/100 g) or physiological saline. Another two groups of rats were fed on laboratory chow with or without 2% DEHP for 2 weeks. Livers were perfusionfixed, cut into tissue sections (50–100 ), and processed to cytochemistry for catalase, immunocytochemistry for SPT, and conventional procedures for electron microscopy. The morphometric analysis showed that glucagon injection has negligible effect on the volume and numerical density and mean diameter of peroxisomes, whereas volume density of mitochondria was decreased by 25%. By DEHP administration peroxisomes were about 3-fold increased in the volume and numerical density. Mitochondria was increased about 40% in the numerical density, but unchanged in the volume density. Light and electron microscopic immunocytochemistry demonstrated that glucagon injection exclusively enhanced mitochondrial SPT, whereas DEHP administration exclusively induced in peroxisomal SPT. Quantitative analysis showed that by the glucagon injection, the labeling density of mitochondria was increased about 4-fold, but that of peroxisomes was 1.6 times as much as control, while by DEHP administration, the labeling density of peroxisomes was enhanced about 3-fold but that of mitochondria was decreased by 13%. The results clearly indicate that glucagon induces mitochondrial SPT, whereas peroxisome proliferator, DEHP induces peroxisomal SPT.     

Neuropeptides in the pineal gland?

Summary In an attempt to localize components of the renin angiotensinsystem in the pineal gland of rats, immunocytochemical studies using the PAP-technique were performed with antisera against angiotensin I, angiotensin II and angiotensinogen. The staining pattern thus obtained was not only the same for the three antisera, but was also identical to that shown for many other peptide-antisera in the literature. In those studies, the immunocytochemical staining had been ascribed to a distinct pineal cell population or to cell processes. However, by examining adjacent semithin and ultrathin sections by immunocytochemistry and electron microscopy, respectively, we could identify the extracellular perivascular compartment and its flocculent material as the site of staining.This unexpected localization and the observation of immunoreactivity of some preimmunsera in the same compartment as well as several additional findings and arguments are taken to suggest the likelihood of pseudopositive immunostaining, typical for the pineal gland.These studies were supported by the German Research Foundation within the SFB 90 Cardivasculäres System     

Immunostaining of growth hormone and prolactin in paraffin-embedded and stored or previously stained materials.

In attempts to evaluate immunocytochemically autopsy and biopsy material previously obtained and processed for conventional histologic staining, we had to resort to immunostaining of tissues embedded years ago or even sections already stained with hematoxylin-eosin or aldehyde thionin-PAS-orange G. Hypophysial growth hormone and prolactin proved remarkably resistant to such prior treatment with regard to their antigenic properties, and could be readily immunostained in tissue embedded in paraffin 3-4 years earlier, and after destaining of sections prepared up to 7 years earlier. The results of such "retrospective" immunocytochemical evaluation of autopsy and biopsy materail is illustrated with the staining of "pregnancy cells" for prolactin in the hypophysis of a woman postpartum, the immunostaining for prolactin in the cells of adenomas associated with marked hyperprolactinemia, the staining for growth hormone in adenomas removed from children with gigantism, and the immunostaining for prolactin, growth hormone or both in several adenomas that were discovered at autopsy and not associated with a known clinical history of endocrine aberrations.     

Immunocytochemistry of 5-bromo-2′-deoxyuridine labelled oligonucleotide probes

Summary A synthetic oligonucleotide probe, complementary to oxytocin m-RNA was labelled enzymatically with 5-bromo-2-deoxyuridine (5-BrdU) and, with [-³²P]-ATP. The labelled probes were used for in situ, hybridization of histological sections of the mouse hypothalamus. A monoclonal antibody to 5-BrdU and the streptavidine-peroxidase technique were used in order to visualize hybridization with the 5-BrdU labelled probe. In situ hybridization with [³²P] labelling was detected autoradiographically. With both methods hybridized neurons were visible in the magnocellular hypothalamic nuclei. While immunostaining and radio-labelling provided similar localization of oxytocin m-RNA, only the immunocytochemical technique showed clear cellular resolution of the reaction product. In situ hybridization with 5-BrdU labelled probes followed by 5-BrdU immunocytochemistry seems to be a powerful alternative to common autoradiographic techniques.     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique

Summary A simple procedure is described for removing Epon resin from semi-thin 1 m sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4–7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.     

Mechanisms of regulation of liver fatty acid-binding protein

Liver fatty acid-binding protein (L-FABP) expression is modulated by developmental, hormonal, dietary, and pharmacological factors. The most pronounced induction is seen after treatment with peroxisome proliferators, which induce L-FABP coordinately with microsomal cytochrome P-450 4A1 and the enzymes of peroxisomal fatty acid -oxidation. These effects of peroxisome proliferators may be mediated by a receptor which has been shown to be activated by peroxisome proliferators in mammalian cell transfection studies. However, the peroxisome proliferators tested thus far do not bind to this receptor, known as the peroxisome proliferator-activated receptor (PPAR), and its endogenous ligand(s) also remain unknown. Peroxisome proliferators inhibit mitochondrial -oxidation, and one hypothesis is that the dicarboxylic fatty acid metabolites of accumulated LCFA, formed via the P-450 4A1 -oxidation pathway, serve as primary inducers of L-FABP and peroxisomal -oxidation. We have tested this hypothesis in primary hepatocyte cultures exposed to clofibrate (CF). Inhibition of P-450 4A1 markedly diminished, via a pre-translational mechanism, the CF induction of L-FABP and peroxisomal -oxidation. In further experiments, long-chain dicarboxylic acids, the final products of the P-450 4A1 -oxidation pathway, but not LCFA, induced L-FABP and peroxisomal -oxidation pre-translationally. These results suggest a role, in part, for long-chain dicarboxylic acids in mediating the peroxisome proliferator induction of L-FABP and peroxisomal -oxidation. We also found that LCFA, which undergo rapid hepatocellular metabolism, could become inducers of L-FABP and peroxisomal -oxidation under conditions where their metabolism was inhibited. The role of the PPAR in mediating these effects is unknown, but clearly warrants further study. The induction of L-FABP and peroxisomal -oxidation by LCFA and/or their -oxidized metabolites may provide a means for limiting the deleterious effects of increased intracellular concentrations of free LCFA, and thus act as an important hepatocellular adaptation to impairment or overload of mitochondrial LCFA oxidation.     

LR-White and LR-Gold resins for postembedding immunofluorescence staining of laminin in mouse kidney

Summary This work describes a method for the immunolocalization of laminin on 1m-thick tissue sections using a postembedding immunofluorescence technique. Embedding of unfixed or formaldehyde-fixed mouse renal cortex in either of the acrylic resins LR-White or LR-Gold permitted reliable postembedding immunofluorescence staining for laminin. LR-White was heat-cured at 50°C whereas LR-Gold was polymerized at –25°C. A stronger immunostaining for laminin was obtained from tissue embedded in polymerized LR-Gold compared with the staining from tissue embedded in LR-White. Prerequisites for adequate postembedding immunostaining are the partial dehydration of the tissue (maximum ethanol concentration, 70%) and pepsin treatment of the tissue sections prior to performing the immunostaining reactions.     

The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution

We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoyfixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some -oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.     

ACTH radioimmunocytochemistry (RICH) on rat anterior pituitary cells

Summary Radioimmunocytochemistry (RICH) was applied to detect corticotrophs in adult rat pituitaries and 8-day-old anterior pituitary monolayers by incubating sections and cultures with ¹²⁵I-ACTH-anti ACTH immune complexes. After incubations autoradiography was made. In comparison, conventional immunostaining was carried out on adjacent sections and parallel cultures. It has been esteblished that RICH is suitable for detection of corticotrophs.     

Proteolysis and lectin histochemistry

Summary Lectin binding to formalin-fixed, paraffin-embedded tissue can often be enhanced by pre-treatment of the sections with proteolytic enzymes. However, the pattern of staining may be profoundly influenced by the type of enzyme preparation which is used.Sites of binding of thirteen different lectins to murine ovary and thyroid gland were studied after exposure of tissue sections to crude trypsin, purified trypsin, purified -chymotrypsin, pepsin, protease VII, papain, bromelain, thermolysin or elastase. With most lectins, the results obtained were similar regardless of which enzyme was used for proteolytic digestion. However, the pattern of binding of soy bean lectin to the ovary and of concanavalin A and common pea lectin to the thyroid gland was highly dependent upon the enzyme used to pre-treat the sections. In both tissues, the staining pattern seen in untreated frozen sections was similar to that found in formalin-fixed, paraffinembedded material digested with purified trypsin, but was different from that observed after exposure of processed sections to crude trypsin. The location of binding sites after treatment of paraffin sections with chymotrypsin was the same as that after digestion with crude trypsin. Results obtained after the use of other proteolytic enzymes varied according to the tissue being studied.These findings imply that the effect of treatment with crude trypsin is due to contaminating chymotrypsin, and demonstrate that the use of purified trypsin may have advantages over other proteolytic enzymes in lectin histochemistry. The observations may also apply to other related cytochemical techniques such as immunocytochemistry.     

Phytanic acid alpha-oxidation and complementation analysis of classical Refsum and peroxisomal disorders

Summary We have measured the production of ¹⁴CO₂ from exogenous [1-¹⁴C] phytanic acid in fibroblast monolayers from patients with classical Refsum's disease and peroxisomal disorders. Activities in the different disorders were (percentage of control): classical Refsum's disease (5%), isolated peroxisomal acyl-CoA oxidase deficiency (75%), Zellweger syndrome (4%), neonatal adrenoleukodystrophy (5%), and rhizomelic chondrodysplasia punctate (3%). Absence of complementation was demonstrated between Zellweger syndrome and infantile Refsum's disease lines after polyethylene glycol fusion, with decreases of average activity of 11% relative to unfused cell mixtures. Classical Refsum's disease, rhizomelic chondrodysplasia punctata, and neonatal adrenoleukodystrophy lines all complemented one another, and Zellweger syndrome or infantile Refsum's disease lines, with average activity increases of 522%–772%. No intragenic complementation was observed within either group. Four complementation groups were detected suggesting that at least four genes are involved in phytanic acid -oxidation: one gene for the enzyme phytanic acid -hydroxylase (probably mitochondrial); one gene for a regulatory factor for the expression of phytanic acid -decarboxylation activity and two membrane-bound peroxisomal enzymes involved in the synthesis of plasmalogens; two genes for the assembly of functional peroxisomes and/or import of proteins into peroxisomes.     

Post-mortem visualization of peroxisomes in rat and in human liver

Summary This paper describes spontaneous post-mortem changes of peroxisomal staining in normal liver and kidney of rats and in human autopsy liver. At room temperature, regional staining loss is observed at 18h after death in rat kidney, at 24h in human liver and at 48 h in rat liver. Preservation at 4°C delays this phenomenon. In human liver, the peroxisomal volume density is decreased at both temperatures at 48 h. After freezing of fresh tissue in dry ice, peroxisomal staining is decreased homogeneously. Under the electron microscope, peroxisomal alterations suggest a loss of catalase activity. These changes do not necessarily preclude the study of peroxisomal features since, even after 48 h at room temperature, peroxisomes are still well stained in the less affected regions. Catalase and three -oxidation enzymes, namely acyl-CoA oxidase, bifunctional protein (with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase) and 3-oxoacyl-CoA thiolase, could be visualized immunocytochemically in human autopsy livers up to 48 h after death. However, the study of certain peroxisomal features such as catalase activity and peroxisomal distribution, may be hampered as the post-mortem period is prolonged.     

Standardization of various applications of methacrylate embedding and silver methenamine for light and electron microscopy immunocytochemistry

Summary The use of butyl-methyl-methacrylate embedding and the application of the silver methenamine (SM) method as a poststaining of the immunoperoxidase-DAB (IP) procedure led to the standardization of several useful methods for the visualization of tissue antigents at the light and electron microscope level. These procedures included: 1) Standardization of the actual methacrylate embedding; 2) The IP-SM method with an without periodic acid oxidation, which provided 100% intensification of the IP staining; 3) The IP-SM method made it possible to stain semithin sections (0.5 m), and this in turn, permitted a) clear visualization under the light microscope of the intracellular distribution of antigens and, b) staining, in several adjacent sections, of roughly the same cytoplasmic region of the same cell with different primary antisera; 4) a double immunostaining whereby the first antigen in the sequence was revealed by the IP-SM method and the second by the IP procedure: 5) standardization of the IP and the IP-SM methods for post-embedding staining of ultrathin methacrylate sections.The combined application of methacrylate embedding and the IP-SM, and the use of an appropriate fixative, resulted in an ultrastructural immunocytochemical procedure characterized by a good immunoreactivity of the tissue sections, a strong and selective immunoreaction and a well preserved ultrastructure.Supported by Grant RS-82-18 from Dirección de Investigaciones, Universidad Austral de Chile, Chile     

Marginal plates in hepatic peroxisomes of Ichthyophis glutinosus (Amphibia: Gymnophiona)

Summary The ultrastructure of hepatic peroxisomes was investigated in Ichthyophis glutinosus (Amphibia: Gymnophiona), employing perfusion fixation and the diaminobenzidine (DAB) technique for the visualization of catalase. The majority of peroxisomes is circular or rod-shaped, although elongated particles occasionally occur. They contain a finely granular matrix, lightly stained after the DAB procedure. Their mean diameter is approximately 0.25 m. Serial sections reveal that the circular and rod-shaped peroxisomal profiles are cross and oblique sections of highly tortuous, tubular organelles exceeding 2 m in length.In addition to tubular profiles, elongated, rectangular particles, as well as straight dumbbell-shaped organelles with distinct marginal plates are observed. They range from 900 to 1650 nm in length (mean = 1200 nm). In the flattened, thin central portion of the dumbbell-shaped particle, the peroxisomal membranes form a cisterna enclosing one or two uniformly thick marginal plates, which display a definite substructure with a periodicity of 10 nm.These findings indicate that peroxisomes in the liver of Ichthyophis exhibit a complex organization. It is suggested that the organelles undergo a specific differentiation process, morphologically characterized by the formation of enlarged segments of unusual shape.This study was supported by grants from the Deutsche Forschungsgemeinschaft, Fa 146/1-2 and Sto 75/9