Light microscopical detection of antigens and lectin binding sites with gold-labelled reagents on semi-thin Lowicryl K4M sections: Usefulness of the photochemical silver reaction for signal amplification

Summary In the present study, we have investigated the applicability of semi-thin sections from low temperature Lowicryl K4M-embedded tissues for cytochemical labelling with protein A—gold and lectin—gold complexes. In order to ensure the best possible signal-to-noise ratio antibodies, protein A—gold and lectin—gold were applied in concentrations used for labelling at the electron microscope level. Furthermore, due to the lack of an appropriate chemical procedure for resin removal, untreated semi-thin sections were incubated. Under such conditions, semi-thin sections displayed either no visible staining or only a faint incomplete staining. However, following photochemical silver reaction, the latent or faint incomplete staining was rendered visible in most cases. It is concluded that the same block of Lowicryl K4M-embedded tissue and the same labelling reagents can be used for both light and electron microscopical cytochemical studies. At the light microscopical level, a high degree of structural and specific staining information is obtained. The reactivity of cellular components with antibodies or lectins is preserved even after years of storage of the blocks or slides containing semi-thin sections.     

Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry

Effect of particle size on labeling intensity in protein A-gold immunocytochemistry was studied. Catalase labeling of rat liver peroxisomes was used as a labeling model. Ultra-thin sections of Lowicryl K4M-embedded rat liver were stained for catalase with protein A-gold (pAg) probes. Five different sizes of colloidal gold probes, from 5 nm to 38 nm in diameter, were prepared. Labeling intensity decreased as the particle size of the pAg probes increased. The highest labeling was obtained by the 5-nm pAg probe and the lowest by the 38-nm pAg probe. Quantitative analysis also showed that labeling density was inversely proportional to the size of gold particles. The results suggest that the pAg probe with small gold particles has high sensitivity.     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.     

Immunocytochemical demonstration of serine:pyruvate aminotransferase in peroxisomes and mitochondria of rat kidney

Summary The light- and electron-microscopic localization of serine:pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Immunocytochemical localization of cathepsins B and H in rat liver

Summary Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultrathin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunocytochemical localization of cathepsins B and H in rat liver

Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Summary Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postomication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Immunocytochemical demonstration of serine: pyruvate amino-transferase in peroxisomes and mitochondria of rat kidney

The light- and electron-microscopic localization of serine: pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of low-temperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry, with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase. The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.     

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postosmication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Prevention of non-specific interactions of gold-labeled reagents on tissue sections

The protein A-gold technique is amongst the most useful labeling techniques available for light and electron microscopic immunolabeling. Some electron microscopic studies, however, have suggested that protein A-gold, and other protein-gold complexes as well, may bind non-specifically to certain tissue structures, particularly in skin, creating a specious pattern of labeling. We utilized the protein A-gold technique with antiserum to both involucrin and keratin under a variety of conditions to document the specificity of labeling. When the standard conditions were followed, the protein A-gold technique produces highly specific results. These conditions include: 1. the blocking of unreacted aldehyde groups by amination; 2. the blocking of non-specific binding sites on tissue sections by preincubation with inert proteins; and 3. the use of proper concentration of the protein A-gold complex. However, non-specific labeling could be produced if the three components of the standard protocol were omitted. In particular, the use of too concentrated protein A-gold lead to non-specific labeling. We report here also updated working protocols for antigen detection with protein A-gold on semithin Lowicryl K4M and paraffin sections which provide optimal staining results.     

Prevention of non-specific interactions of gold-labeled reagents on tissue sections

Summary The protein A-gold technique is amongst the most useful labeling techniques available for light and electron microscopic immunolabeling. Some electron microscopic studies, however, have suggested that protein A-gold, and other protein-gold complexes as well, may bind non-specifically to certain tissue structures, particularly in skin, creating a specious pattern of labeling.We utilized the protein A-gold technique with antiserum to both involucrin and keratin under a variety of conditions to document the specificity of labeling. When the standard conditions were followed, the protein A-gold technique produces highly specific results. These conditions include: 1. the blocking of unreacted aldehyde groups by amination; 2. the blocking of non-specific binding sites on tissue sections by preincubation with inert proteins; and 3. the use of proper concentration of the protein A-gold complex. However, non-specific labeling could be produced if the three components of the standard protocol were omitted. In particular, the use of too concentrated protein A-gold lead to non-specific labeling.We report here also updated working protocols for antigen detection with protein A-gold on semithin Lowicryl K4M and paraffin sections which provide optimal staining results.Part of this work was presented at the 17th World Congress of Dermatology, Berlin (West), May 24–29, 1987     

The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.     

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Immunocytochemical localization of cathepsin H in rat kidney. Light and electron microscopic study

Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For light microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Comparison of the pattern of expression of Leu-M1 antigen in adenocarcinomas,neutrophils and Hodgkin's disease by immunoelectron microscopy

The mouse monoclonal antibody anti-Leu-M1 (CD15) recognizes the carbohydrate determinant lacto-N-fucopentaose III, an oligosaccharide believed to be involved in cell-cell interactions. Anti-Leu-M1 is used in surgical pathology as an aid in the diagnosis of Hodgkin's disease. Additionally, adenocarcinomas derived from various organs stained positively with anti-Leu-M1 at the light microscopic level. Since mesotheliomas do not display positive reactivity to this antibody, Leu-M1 is clinically useful as part of a panel of antibodies in distinguishing adenocarcinomas from mesotheliomas. Previous work was carried out using post-embedding protein A-gold immunocytochemistry on thin sections embedded in Lowicryl K4M from a patient with Hodgkin's disease of the nodular sclerosing type; intense and precise labeling by gold particles was revealed in cytoplasmic granules, which were often clustered in a perinuclear location, in the Golgi apparatus, and focally along the plasma membrane of Reed-Sternberg cells. Moreover, polymorphonuclear leukocytes demonstrated similar labelling along the plasma membrane and over cytoplasmic granules. To define precisely the intracellular localization of Leu-M1 in human adenocarcinomas, we have performed post-embedding immunoelectron microscopy with the protein A-gold technique on sections embedded in Lowicryl K4M from neoplasms of the lung, stomach, colon, and breast. The pattern of labeling by gold particles indicative of Leu-M1 binding varied in adenocarcinomas of the different organs.     

Localization of urate oxidase in the crystalline cores of rat liver peroxisomes by immunocytochemistry and immunoblotting

We investigated the immunocytochemical localization of urate oxidase by light and electron microscopy. Rabbits were immunized with urate oxidase prepared from rat liver and the resulting antibody was further purified by affinity chromatography. Immunoblotting of the antigen revealed a single band of Mr 32,500 daltons, consistent with a subunit of uricase. The same band was observed in immunoblots prepared from a total peroxisome fraction and in its subfraction containing the cores, but not in the matrix portion. Immunostaining of 1-micron sections with the antibody against uricase followed by protein A-gold-silver showed fine granules in hepatocytes, which exhibited distinct fluorescence when examined in a microscope equipped with epifluorescence illumination. Incubation of ultra-thin sections of rat liver, embedded in Lowicryl K4M, LR White, or Epon, with the anti-uricase antibody followed by protein A-gold showed prominent labeling of the crystalline cores, with no reaction in the surrounding peroxisomal matrix. In contrast, the core region was spared whereas the matrix was heavily labeled in sections incubated with an antibody against catalase. Direct incubation of cores, isolated by centrifugation, with the anti-uricase antibody followed by protein A-gold revealed gold particles on the surface of isolated cores, with rare particles within the lumen of the polytubular structures that make up the cores. Specificity of the immunolabeling was established in sections incubated with an IgG fraction from pre-immunized rabbits. These observations demonstrate that in normal rat liver urate oxidase is exclusively associated with the crystalline cores in peroxisomes.     

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a ³⁵S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascinspecific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.