The distribution of la antigens of the H-2 complex on lymph node cells by immunoferritin labelling

The distribution of Ia antigens on the surfaces of lymph node lymphocytes of several mouse strains was investigated using indirect immunoferritin labeling and electron microscopy. The immunoferritin labeling results agreed with results of cytotoxic tests in strain distribution of reactivity, proportion of cells showing label, and cell populations reacting. Capping was induced by increased incubation temperature but conditions for Ia antigen mobilization varied somewhat between the two anti-Ia antisera employed. Uncapped specimens generally showed a denser, more evenly distributed antigen coating than is the case for H-2 antigens labeled by the same indirect immunoferritin method.     

Xiphinema index and Caenorhabditis elegans: preparation and molecular labeling of ultrathin frozen sections

A method for the orientation of nematodes for cryoultramicrotomy is described. Comparison of cryosections with sections prepared by conventional electron microscopic procedures showed satisfactory resolution of structural details in frozen sections. Labeling of frozen sections en face was achieved by cationized ferritin and colloidal iron. Actin was localized in cryosections of somatic muscle by immunoferritin labeling. The current study is a practical example of the application potential of cryoultramicrotomy to examination of nematode cytochemistry at a molecular level.     

Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes

Synapsin I (protein I) is a major neuron-specific endogenous substrate for cAMP-dependent and Ca/calmodulin-dependent protein kinases that is widely distributed in synapses of the central and peripheral nervous system (De Camilli, P., R. Cameron, and P. Greengard, 1983, J. Cell Biol. 96:1337-1354). We have now carried out a detailed analysis of the ultrastructural localization of synapsin I in the synapse. For this purpose we have developed a novel immunocytochemical technique that involves the labeling of isolated synaptosomes immobilized in a thin agarose gel. Special fixation conditions were designed to maximize accessibility of synapsin I to marker molecules. Immunoferritin and immunoperoxidase studies of this preparation indicated that synapsin I is localized in the presynaptic compartment and that it is present in close to 100% of all nerve endings. Immunoferritin labeling also indicated that, inside the nerve ending, synapsin I is specifically associated with the cytoplasmic surface of synaptic vesicles. In agreement with these immunoferritin results, the labeling produced by immunoperoxidase was compatible with a specific association of synapsin I with synaptic vesicle membranes. However, at variance with the very specific distribution of immunoferritin, immunoperoxidase reaction product was also found on other membranes of the terminals, presumably as a result of its diffusion over a short distance from the synaptic vesicles. Anti-synapsin I immunoperoxidase staining of tissue sections for electron microscopy produced an uneven labeling of terminals of the neuropile, in agreement with results of a previous study (Bloom, F. E., T. Ueda, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA. 76:5982-5986). A comparison with results obtained in isolated synapses indicates that the limited labeling of nerve endings in tissue sections results from limited and uneven penetration by marker molecules. The specific association of synapsin 1 with synaptic vesicle membranes in the great majority of nerve terminals suggests a prominent role for this phosphoprotein in the regulation of synaptic vesicle function.     

TdTomato and EGFP identification in histological sections: insight and alternatives

Abstract

Tandem dimer Tomato (tdTomato) provides a useful alternative to enhanced green fluorescent protein (eGFP) for performing simultaneous detection of fluorescent protein in histological sections together with fluorescence immunohistochemistry (IHC). eGFP has many properties that make it useful for cell labeling; however, during simultaneous fluorescence IHC, the usefulness of eGFP may be limited. This limitation results from a fixation step required to identify eGFP in histological tissue sections that can mask antibody epitopes and adversely affect staining intensity. An alternative fluorescent protein, tdTomato, may assist concurrent detection of fluorescent protein within tissue sections and fluorescence IHC, because detection of tdTomato does not require tissue fixation. Tissue sections were obtained from various organs of mice ubiquitously expressing eGFP or tdTomato that were either unfixed or fixed with 4% paraformaldehyde. These tissues later were combined with fluorescence IHC. Both eGFP and tdTomato displayed robust signals in fixed frozen sections. Only tdTomato fluorescence, however, was detected in unfixed frozen sections. Simultaneous detection of fluorescence IHC and fluorescent protein in histological sections was observed only in unfixed frozen tdTomato tissue. For this reason, tdTomato is a useful substitute for eGFP for cell labeling when simultaneous fluorescence IHC is required.     

Disposition of exposed antigens on the faces of isolated Mycoplasma gallisepticum membranes.

The transverse disposition of exposed protein antigens on the two faces of isolated Mycoplasma gallisepticum membranes have been investigated by using indirect immunoferritin labeling to accomplish visualization of the antigens at the ultrastructural level. Comparison between the labeling patterns obtained with unabsorbed specific mycoplasma antiserum and antiserum from which antibodies directed against outer side determinants had been removed revealed that the majority of protein antigens were the same on the opposed membrance faces or at least displayed extensive interside cross-reactivity. The relatively scarce tagging of isolated Acholeplasma laidlawii membranes, contrary to membranes on intact organisms observed in this investigation, precluded conclusions regarding the disposition of membrane antigens of this species. The advantages and limitations of the employed method in disposition studies and the factors influencing the transverse distribution of membrane proteins in mycoplasmas are discussed.     

Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections.

We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections.     

Effect of fixation and epitope retrieval on BrdU indices in mammary carcinomas.

Studies in which 5-bromo-2'-deoxyuridine (BrdU) is used to quantify rates of cell proliferation are conducted prospectively. Therefore, the opportunity exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU epitope was masked by formalin vs methacarn fixation in the assessment of cell proliferation. Mammary carcinomas from animals pulse-labeled with BrdU were trisected. A portion was frozen and the remaining two portions were fixed in 10% neutral buffered formalin or methacarn for 24 hr, processed, embedded in paraffin, and sections stained for incorporated BrdU using a peroxidase immunohistochemical staining technique. Antigen retrieval techniques also were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in unfixed frozen sections (17.5%). Formalin fixation alone dramatically suppressed the labeling index (0.3%), which was only partially recovered using various antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recommended for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.     

The occurrence and distribution of H-2 antigens on mouse intestinal epithelial cells.

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from the small intestine by EDTA and trypsin. Before cell dissociation, the intestine was prefixed in paraformaldehyde or periodate-lysine-paraformaldehyde in order to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. The results demonstrated the presence of H-2 antigens on the lateral and basal cell membranes at about the same high density that was observed at the surface of mouse monocytes. No H-2 antigens could be detected at the apical surface of dissociated or undissociated epithelial cells. It is unlikely that the fuzzy coat masked H-2 antigens at the apical surface because it was essentially absent from the apical membranes of dissociated cells. These observations extend our knowledge of the cellular distribution of transplantation antigens, and provide further evidence of a discontinuity in the expression of membrane components at the junctional complex of epithelial cells.     

Ultrastructural localization of calsequestrin in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections

The ultrastructural localization of calsequestrin in rat skeletal muscle (gracilis) was determined by indirect immunoferritin labeling of ultrathin frozen sections. Calsequestrin was found in the lumen of transversely and longitudinally oriented terminal cisternae but was absent from most of the longitudinal sarcotubules and the fenestrated sarcoplasmic reticulum. Calsequestrin was occasionally observed in vesicular structures found in the central region of the I band. Since calsequestrin is believed to provide the major site of Ca2+ sequestration in the sarcoplasmic reticulum, the present results support the view that Ca2+, transported to the lumen of the sarcoplasmic reticulum, is preferentially sequestered in the terminal cisternae, but they also suggest that additional Ca2+ sequestration may occur near the center of the I band.     

Optimal antigen localization in human tissues using aldehyde-fixed plastic-embedded sections

Although the utility of antigen labeling techniques in frozen tissues is well known, it is generally acknowledged that an improvement in morphologic preservation is desirable. Conventionally processed paraffin-embedded tissues are limited in the range of antigens that can be detected and newer plastic embedding techniques have been even more restricted. By using cold (4 degrees C) processing and limited fixation a wide range of antigens (including T and B markers) has been demonstrated in 2 mu plastic sections. The morphologic preservation and antigen localization are superior to other techniques. The combination of precise antigen localization and excellent morphologic preservation should expand the diagnostic and investigative uses of immunohistology.     

Immunocytochemical localization of the main intrinsic polypeptide (MIP) in ultrathin frozen sections of rat lens

The in situ distribution of the 26-kdalton Main Intrinsic Polypeptide (MIP or MP 26), a putative gap junction protein in ocular lens fibers, was defined at the electron microscope level using indirect immunoferritin labeling of ultrathin frozen sections of rat lens. MIP was found distributed throughout the plasma membrane of the lens fiber cell, with no apparent distinction between junctional and nonjunctional membrane. MIP was not detectable in the basal or lateral plasma membrane of the lens epithelial cell, including the interepithelial cell gap junctions; nor was MIP detectable in the plasma membrane or gap junctions of the hepatocyte. Previous reports have indicated that the protein composition of the lens fiber cell junction differs from that of the hepatocyte gap junction. The evidence presented here suggests that the composition of the fiber cell junction and plasma membrane is also immunocytochemically distinct from that of its progenitor, the lens epithelial cell.     

Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study

Amylase (Am) and chymotrypsinogen (Chtg) were demonstrated in rat and guinea pig exocrine pancreatic cells by immunofluorescence and immunoferritin cytochemistry on thin and ultrathin frozen sections. We describe two observations indicating that Am and Chtg may behave differently in the pre-Golgi phase of their intracellular transport. Firstly, aggregates of material within the RER cisternae of the guinea pig (so-called intracisternal granules) reacted strongly with anti-Chtg, but showed no affinity for anti-Am. Secondly, in both rat and guinea pig, the increase in labeling intensity from cytoplasm (RER) to secretory granules was larger for Chtg than for Am. We hypothesize that the two proteins do not travel in-parallel towards the Golgi complex. Compared with Chtg, Am would lag behind in the RER cisternae.     

Tissue preparation for the demonstration of surface antigens by immunoperoxidase techniques

Summary The fixation and drying regimes for frozen sections and cytocentrifuge preparations for the demonstration of surface antigens, such as immunoglobulins and iron binding proteins, vary enormously between different groups of workers. A method using freeze-dried sections and acetone fixation was compared with 16 other methods of fixation and found to be the best for tissue preservation and antigen demonstration. Freeze drying was found to improve the cytological preservation of air-dried sections considerably.     

Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex

Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.     

A new fixed cryosection technique for the simultaneous immunocytochemical demonstration of T6 and S100 antigens

Summary A formalin—calcium fixation method of preparing cryosections is described, which allows demonstration of Langerhans' cells by S100 antigen staining on frozen sections. The number of Langerhans' cells given by T6 antigen staining is also higher in formalin—calcium fixed frozen sections than acetone fixed frozen sections. The preparation is suitable for dual demonstration of the two antigens on the same section enabling a more accurate numerical evaluation of Langerhans' cell populations in the normal cervical epithelium.     

A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabeling.

Ultracryotomy of fixed specimens in combination with immunogold labeling is widely used for ultrastructural localization of many interesting molecules. Since the introduction of this technique, vast improvements in techniques and machinery have been established and the entire process has been made easier and more accessible. Normally, sections are cut and labeled within 1 day to prevent possible loss or redistribution of soluble antigens within the sections. An increasing demand for more sections and multiple labeling protocols prompted us to investigate the extent to which ultrathin cryosections can be stored. This would render the time spent behind an ultracryomicrotome more efficient and would allow immunogold labeling at a later stage. We investigated whether gelatin plates, 2.3 M sucrose, or 1.0% methyl cellulose/1.2 M sucrose can be used to store thawed frozen sections for a longer period of time. Ultrathin sections of mildly fixed tissue and cultured cells were stored for up to 6 months before immunogold labeling. The preservation of the ultrastructure of stored sections was excellent and was similar to that of immediately processed sections. Importantly, prolonged storage did not affect the labeling intensity.     

LR White is preferable to Unicryl for immunogold detection of fixation-sensitive nuclear antigens

The purpose of this study was to compare two electron microscopy embedding media - LR White and Unicryl - with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation.     

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)     

Fixation, processing, and immunochemical reagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue

Fixatives, fixation additives, paraffin processing reagents, and immunochemical reagents were investigated for effects on preservation of T-lymphocyte surface membrane antigens CD3, CD4, and CD8 in human tonsil. Individual reagent effects were assessed in frozen sections by use of monoclonal antibodies and this information was used to optimize T-cell immunostaining in paraffin sections. Harmful factors were fixation delay, fixation at acid pH, fixation and processing at temperatures above 4 degrees C, hot paraffin wax, proteolytic enzymes, methanolic hydrogen peroxide, Triton X-100, and prolonged iodine treatment. Optimal T-cell demonstration in paraffin sections followed tissue fixation in periodate-lysine-paraformaldehyde dichromate at 4 degrees C, pH 7.5; processing through isopropanol, then xylene or chloroform, at 4 degrees C; and embedding in low melting point wax at 45-50 degrees C. Graded antigen stability occurred: CD3 most stable, CD8 least, and CD4 intermediate. CD4 and CD8 antigen preservation in paraffin sections required critical optimal tissue handling. CD3 was more stable and was also demonstrated in tissue fixed in commercial formalin, glutaraldehyde, and Bouin's fluid when fixation and processing conditions were optimized for pH and temperature. Of the fixation additives studied, polyethylene glycol and several potassium and magnesium salts enhanced immunostaining, whereas calcium chloride and lidocaine were deleterious.     

Ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections

The ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat gracilis muscle was determined by indirect immunoferritin labeling of ultrathin frozen sections. Simultaneous visualization of ferritin particles and of adsorption- stained cellular membranes showed that the Ca2+ + Mg2+-ATPase was concentrated in the longitudinal sarcoplasmic reticulum and in the nonjunctional regions of the terminal cisternae membrane but was virtually absent from mitochondria, plasma membranes, transverse tubules, and junctional sarcoplasmic reticulum. Ferritin particles were found preponderantly on the cytoplasmic surface of the membrane, in agreement with published data showing an asymmetry of the Ca2+ + Mg2+- ATPase within the sarcoplasmic reticulum membrane. Comparison of the density of ferritin particles in fast and slow myofibers suggested that the density of the Ca2+ + Mg2+-ATPase in the sarcoplasmic reticulum membrane in a fast myofiber is approximately two times higher than in a slow myofiber.