Immunocytochemical localization of carbonic anhydrase on ultrathin frozen sections with protein A-gold

Summary The protein A-gold technique was used to localize carbonic anhydrase isozymes on ultrathin frozen sections of kidney collecting duct epithelial cells and erythrocytes. The particulate nature of the gold marker gives a more precise appreciation of the intracellular distribution of this enzyme than has been previously possible, and allows the intensity of the labeling to be quantified. Intercalated cells showed four times more labeling over the cytosol than adjacent principal cells in collecting ducts from the inner stripe of the outer medulla: by double-labeling using protein A-gold particles of different sizes, carbonic anhydrase isozymes B and C were simultaneously localized in erythrocytes.     

Immunocytochemical localization of carbonic anhydrase on ultrathin frozen sections with protein A-gold

The protein A-gold technique was used to localize carbonic anhydrase isozymes on ultrathin frozen sections of kidney collecting duct epithelial cells and erythrocytes. The particulate nature of the gold marker gives a more precise appreciation of the intracellular distribution of this enzyme than has been previously possible, and allows the intensity of the labeling to be quantified. Intercalated cells showed four times more labeling over the cytosol than adjacent principal cells in collecting ducts from the inner stripe of the outer medulla: by double-labeling using protein A-gold particles of different sizes, carbonic anhydrase isozymes B and C were simultaneously localized in erythrocytes.     

Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry

The fluorescent carbocyanine dye DiI can be used for retrograde and anterograde labeling of neuronal pathways. To investigate the possible neurochemical identity of DiI-labeled neuronal cell bodies and terminals, we used a procedure for double-labeling of the same tissue with antisera to specific neuroactive substances. This procedure involves visualizing the immunohistochemical label with an FITC-conjugated secondary antiserum. Both labels can be viewed in the same tissue by fluorescence microscopy, and individual cell bodies and processes double-labeled with DiI and antiserum can be identified by switching between filter sets appropriate for rhodamine (to see the DiI labeling) and for fluorescein (to see the immunhistochemical labeling). The method has been used with primary antisera to excitatory and inhibitory amino acid neurotransmitters, as well as to neuropeptides, and is likely to be useful with antibodies against a wide variety of substances. Several other immunocytochemical methods were found to be incompatible with DiI labeling.     

Cellular and subcellular distribution of the calcium-binding protein NCS-1 in the central nervous system of the rat

NCS-1 (neuronal calcium sensor) is a recently characterized member of a highly conserved neuron-specific family of calcium-binding proteins, which also includes frequenin and recoverin. The cellular and subcellular distributions of NCS-1 in the rat nervous system were investigated using light- and electron-microscopic immunohistochemistry. NCS-1 immunoreactivity was localized to neuronal cell bodies and axons throughout the brain and spinal cord but not to glial cells. The most intense labeling was observed in myelinated axons, the axonal ramifications of the basket cell in the cerebellar cortex, and large neurons in the brainstem and pons. These same structures were also characterized by heavy labeling for neurofilament protein, as determined by double-labeling experiments. Most axon terminals were unlabeled or only lightly labeled. The most remarkable subcellular staining occurred in the perikarya where intense labeling was associated with the membranes of the trans saccules of the Golgi apparatus. The widespread distribution of NCS-1 indicates that it may be active in a variety of calcium-dependent neuronal functions, whereas the specific subcellular localization to the Golgi apparatus and neurofilament-rich structures suggests a specialized role in calcium regulated protein trafficking and cytoskeletal interactions.     

Proteolytic 18O labeling for comparative proteomics: evaluation of endoprotease Glu-C as the catalytic agent

Recently, proteolytic 18O labeling has been demonstrated as a promising strategy for comparative proteomic studies (Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-42). In this approach, protein mixtures are digested in parallel in H216O and H218O and the ratios of isotopically distinct peptide products are measured by mass spectrometry. In the initial report from this laboratory, trypsin was shown to catalyze incorporation of two 18O atoms into the carboxyl terminus of each new peptide formed by cleavage of the adenovirus proteome. In the present study, a second enzyme, endoprotease Glu-C, is evaluated as an agent for cleavage and labeling. Proteolytic 18O labeling by Glu-C is shown to occur readily with phosphorylated and glycosylated proteins and with cysteinealkylated and disulfide-linked proteins. A sequential double-labeling strategy is used to characterize N-linked glycopeptides. Labeled and unlabeled peptide pairs are found to coelute chromatographically, and measurements of isotope ratios by nanospray and capillary LC-MS are found to be accurate and precise.     

Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis

Direct immunogold labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a "control" tissue to identify preparation and labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during labeling, and several factors limiting quantitative analysis.     

Preservation and Long-Term Storage of Feulgen-Stained Mouse Tissues for Subsequent Autoradiography

Tissue of the jejunal crypts of mouse intestine was fixed for 24-48 hr in acetic-ethanol, 1:3, and stained en bloc by the Feulgen reaction. The stained preparations were then stored 4 mo at -25 C in either 45% acetic acid alone or in dimethyl sulfoxide (DMSO) or glycerol to which 45% acetic acid had been added to make 15% of the total volume. Such storage preserved not only the stain but allowed autoradiographs to be made. No loss of silver grains or a decrease of labeling index was observed. The procedures are equally successful when used with double-labeling experiments. Solid, transplantable, experimental carcinomas can be preserved in a manner identical to that suggested for the epithelial cells of the crypts.     

The phosphatidylinositol transfer protein in 3T3 mouse fibroblast cells is associated with the Golgi system.

By use of indirect immunofluorescence it was shown that the phosphatidylinositol transfer protein (PI-TP) in 3T3 mouse fibroblast cells is associated with the Golgi system. This was concluded from double-labeling experiments with TRITC-labeled Ricin which binds to sugar residues that are specifically processed in the Golgi system. Independent evidence for this association was provided by the fact that dissociation of the Golgi system by brefeldin A was reflected in an extensive redistribution of PI-TP labeling. In addition, PI-TP is localized in the cytoplasm and in the nucleus. In exponentially growing cells an enhanced labeling of PI-TP was observed in the cytosol and in the cytosol and in the Golgi system in comparison with quiescent cells. By Western blot analysis and by transfer activity assays, it was confirmed that the concentration of PI-TP was increased in exponentially growing cells. These results strongly suggest that PI-TP fulfills a role in the functioning of the Golgi complex.     

The Effect of Hydrocortisone on the Epidermal Proliferation in an Organ Culture of Chick Embryonic Skin

Hydrocortisone is regarded as an initiator of keratinization in embryonic skin. The present investigation dealt with the effect of hydrocortisone on the proliferation of epidermal cells during early development: Cell kinetic analyses using ³H-thymidine autoradiography were applied to a skin organ culture prepared from a 13-day chick embryo.
Hydrocortisone at a concentration between 0.01 and 1.0 μg/ml was effective in initiating a morphological change leading to the epidermal keratinization in vitro and caused a marked decrease in the mitotic and labeling indices of epidermal basal cells, the decrease being maximum at 2 days of culture previous to the morphological change.
During continuous labeling with ³H-thymidine, the number of labeled basal cells reached 100% within 2 days in the control and 4 days in the culture treated with hydrocortisone. This confirmed that the growth fraction of epidermal basal cells was 1.0 even after the administration of hydrocortisone.
The duration of each cell cycle phase at 2 days of culture was determined by percent labeled mitoses and double-labeling analyses. It was concluded that hydrocortisone extended the generation time of epidermal basal cells at this time point about three fold over the control. This extension was mainly due to the elongation of the G ₁ phase.     

Endocytic pathways and time sequence of lysosomal transfer of macromolecules in cultured mouse peritoneal macrophages

Summary A double-labeling protocol was used to study endocytic pathways and lysosomal transfer of exogenous macromolecules in cultured mouse peritoneal macrophages. After pulse-chase labeling of lysosomes with horseradish peroxidase (visualized cytochemically), the cells were exposed to native, anionic ferritin for 0–45 min at 37° C and then analysed by transmission electron microscopy. The results show that ferritin binds to the plasma membrane, accumulates in coated pits, and is rapidly taken up in small, smooth-surfaced endocytic vesicles. The latter carry the ferritin molecules directly to lysosomes, recognized by their peroxidase labeling, or fuse with each other to form larger endocytic vacuoles (endosomes) which in turn fuse with and empty their content into lysosomes. The first signs of transfer of ferritin into the lysosomes were seen after 5–10 min of exposure and after 25–30 min most of the lysosomes were labeled. Union of ferritin-labeled and other lysosomes was also noted, suggesting that the contents of the lysosomes were spread within the lysosomal compartment by fusion-fission processes. It is concluded that a multiplicity of structures is involved in the uptake and intracellular transport of exogenous macromolecules in macrophages and that the time sequence of lysosomal transfer of the interiorized material is highly variable.     

Strategy for efficient site-specific FRET-dye labeling of ubiquitin

To study conformational changes within a single protein molecule, sp-FRET (single pair fluorescence resonance energy transfer) is an important technique to provide distance information. However, incorporating donor and acceptor dyes into the same protein molecule is not an easy task. Here, we report a strategy for the efficient double-labeling of a protein on a solid support. An ubiquitin mutant with two Cys mutations, one with high solvent accessibility and the other with low solvent accessibility, was constructed. The protein was bound to magnetic beads and reacted with the dyes. The first dye reacted with the side-chain of the Cys with the high solvent accessibility and the second with the other Cys under partially denaturing conditions. Using this method, we can easily label two dyes in a site-specific way on ubiquitin with a satisfied yield. The labeling sites for donor and acceptor dyes can be easily swapped.     

New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP

In vivo green fluorescent protein (GFP)/red fluorescent protein (RFP) double-labeling studies have been hampered by several inconvenient properties of DsRed, the first described RFP. These disadvantages include a very slow (> 24 h) maturation time, emission of contaminating green light, and low solubility. A recently developed variant of DsRed, called DsRed.T4, has a much shorter maturation time, no significant green emission, and improved solubility. We have constructed Drosophila P-element transformation vectors encoding DsRed.T4 for promoter/enhancer analysis, labeling of living cells, or RFP tagging of proteins. These new vectors have all of the features of the widely used Pelican/Stinger GFP vectors, including insulator sequences to reduce position effects, an extensive polylinker, and both cytoplasmic and nuclear-localized forms of the reporter. We have also constructed an upstream activating sequence (UAS)-DsRed.T4 vector, for GAL4 activation of the reporter. We find that DsRed.T4 is very easily detected in transgenic flies without contamination of the GFP signal and that it matures to its fluorescent form nearly simultaneously with GFP. This advance in Drosophila reporter technology makes timed double-labeling experiments in developing transgenic animals possible for the first time.     

Functional interaction of caveolin-1 and eNOS in myocardial capillary endothelium revealed by immunoelectron microscopy.

Immunogold labeling on samples of isolated perfused rat hearts embedded by an innovative low-temperature LR White procedure provided detailed insight into the interaction of caveolin-1 and endothelial NOS in myocardial capillary endothelium at the subcellular level. Separately, the localization of caveolin-1 and eNOS at caveolae under steady state conditions was visualized. A double-labeling experiment supported their close co-localization. Short-term bradykinin stimulation caused a detectable dissociation of eNOS from caveolin and its redistribution to different cell compartments, whereas caveolin itself remained stationary at caveolae. Morphometric analysis revealed that more than 80% of detectable eNOS was co-localized with caveolin-1 at caveolae under control conditions. After brief stimulation for 2 min with 10(-7) M bradykinin, only 26% of the eNOS signals were associated with caveolin-1 and randomly distributed over the endothelial cells. After stimulation, eNOS was found at the plasmalemmal and intracellular membranes, freely in the cytoplasm, and at outer mitochondrial membranes.     

Multiple epitope labeling by the exclusive use of alkaline phosphatase conjugates in immunohistochemistry

 Here we demonstrate a simple and reliable multiple epitope labeling technique based exclusively on the alkaline phosphatase (AP) enzyme-linked visualization method. AP is functionally blocked by ethylenediaminetetraacetic acid (EDTA), which allows the repeated use of AP conjugates in immunohistochemistry with different substrates. We found that reactivation of AP function following EDTA incubation is dependent on EDTA concentration and incubation time. While incubation times of up to 2 h in 0.25 M EDTA, pH 6, exhibit a resumption of AP enzyme function, more than 2 h of incubation irreversibly blocks AP enzyme activity. Surprisingly, EDTA incubation also results in considerable but not complete inhibition of antibody crossreactivity during immunohistochemistry. Thus, this technique is suitable for single-layer, multiple-staining experiments with AP-linked primary antibodies or multilayer labeling with antibodies of various species for sequential staining rounds. We demonstrate the applicability of this technique in immunohistochemistry by double-labeling experiments using the monoclonal antibodies anti-glial fibrillary acidic protein, anti-leucocyte common antigen, anti-CD43/CD45RA (pan-human leucocyte), and anti-migration inhibitory factor-related protein-8 in combination with an in situ nick translation assay to characterize differentiating antigens of apoptotic cells in human glioblastoma paraffin sections. Accepted: 2 April 1998     

Ultrastructural localization of the mannose 6-phosphate receptor in rat liver

An affinity-purified rabbit antibody against rat liver mannose 6- phosphate receptor (MP-R) was prepared. The antibody was directed against a 215 kd-polypeptide and it recognized both ligand-occupied and free receptor. Anti-MP-R was used for immunofluorescence and immunoelectron microscopy of cryosections from rat liver. MP-R was demonstrated in all parenchymal liver cells, but not in endothelial lining cells. MP-R labeling was found at the entire plasma membrane, in coated pits and coated vesicles, in the compartment of uncoupling receptor and ligand, and in the Golgi complex. Lysosomes showed only scarce MP-R label. In double-labeling immunoelectron microscopy, MP-R co-localized with albumin in the Golgi cisternae and in secretory vesicles with lipoprotein particles. Cathepsin D was associated with MP- R in the Golgi cisternae. This finding indicates that MP-R/cathepsin D complexes traverse the Golgi complex on their way to the lysosomes. The possible involvement of CURL in lysosomal enzyme targeting is discussed.     

Novel Formula for Cell Kinetics in Xenograft Model of Hepatocellular Carcinoma Using Histologically Calculable Parameters

The growth rate of tumors should be assessed in terms of both tumor cell proliferation and death. The former is considered to be determined by growth fraction and cell-cycle time, whereas the latter is mainly determined by apoptosis, especially in tumors with a low level of necrosis. While most hepatocellular carcinomas (HCCs) in a relatively early stage contain only a small amount of necrosis, the growth rate supposedly depends mainly on growth fraction, cell-cycle time, and apoptosis. However, their quantitative relationship remains unknown. We have derived a novel theoretical formula for determining this relationship in nonnecrotic HCC, using Ki-67-positive index, apoptotic score, and a correction factor, all calculable by histological assessment without injecting labeling agents. Furthermore, we confirmed the reliability of this formula, using a xenograft model of human HCC with less than 15% necrosis. In this model the values of cell-cycle time calculated from the formula were very close to those estimated by a conventional double-labeling method and showed high correlations. Since our novel formula can clarify the cell kinetics without cumbersome labeling procedures, it is expected to be clinically applicable to HCC with a small portion of necrosis, using the radiographically measured growth rate and the histologically assessed cell kinetic parameters.     

Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.     

Novel application of tyramide signal amplification (TSA): ultrastructural visualization of double-labeled immunofluorescent axonal profiles.

Fluorescent immunocytochemistry (FICC) allows multiple labeling approaches when enzyme-based techniques are difficult to combine, such as in double-labeling experiments targeting small-caliber axonal segments. Nevertheless, the conversion of FICC to a product visible at the electron microscopic (EM) level requires labor-intensive procedures, thus justifying the development of more user-friendly conversion methods. This study was initiated to simplify the conversion of FICC to EM by employing the unique properties of tyramide signal amplification (TSA), which allowed the simultaneous targeting of a fluorescent tag and biotin label to the same antigenic site. Briefly, one of two antigenic sites typically co-localized in damaged axonal segments was visualized by the application of a fluorescent secondary antibody, with the other tagged via a biotinylated antibody. Next, an ABC kit was used, followed by the simultaneous application of fluorophore-tyramide and biotin-tyramide. After temporary mounting for fluorescent digital photomicroscopy, sections were incubated in ABC and reacted with diaminobenzidine before EM analysis. Double-labeling fluorescent immunocytochemistry with TSA clearly delineated damaged axonal segments. In addition, these same axonal segments yielded high-quality EM images with discrete electron-dense reaction products, thereby providing a simple and reproducible means for following fluorescent analysis with EM.     

Covalent structure of mutacin 1140 and a novel method for the rapid identification of lantibiotics.

The primary structure of the Streptococcus mutans lantibiotic mutacin 1140 was elucidated by NMR spectroscopy, mass spectrometry, and chemical sequencing. The structure is in agreement with other closely related lantibiotics, such as epidermin. A novel method was developed in which mutacin 1140 was chemically modified with sodium borohydride followed by ethanethiol, allowing the differentiation of the thioether-containing residues from the dehydrated residues. This double-labeling strategy provides a simple method to reliably identify all modified lantibiotic residues with a minimal amount of material. While NMR spectroscopy is still required to obtain thioether bridging patterns and thus the complete covalent structure, the double-labeling technique, along with mass spectrometry, provides most of the information in a fraction of the time required for a complete NMR analysis. Thus, with these new techniques lantibiotics can be rapidly characterized.     

Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands.

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine system.