Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell.

The location of the cytadherence-accessory high-molecular weight proteins 1 and 4 (HMW1/4) within Mycoplasma pneumoniae cells has been studied by both biochemical and electron microscopic techniques. Peptide mapping studies demonstrated that HMW1/4 share almost identical peptide profiles, suggesting that the two proteins are structurally related. Examination of thin sections of M. pneumoniae with antibodies to HMW1/4 and colloidal gold particles revealed distinct labeling of the filamentous extensions of the mycoplasma cells. Labeling was absent on thin sections of a cytadherence-deficient variant lacking HMW1/4. HMW1/4 partitioned in the detergent-insoluble fraction following Triton X-100 extraction, and analysis by sucrose density gradient centrifugation suggested that HMW1/4 are part of a high-molecular-weight, multiprotein complex. These results were confirmed by immunogold labeling of Triton X-100-extracted M. pneumoniae cells incubated with antibodies to HMW1/4: gold particles bound in specific clusters to detergent-insoluble filaments. Finally, immunogold labeling of whole cells revealed that HMW1/4 are exposed on the cell surface, although to a lesser degree than on the cell interior. These findings indicate that HMW1/4 are membrane proteins associated with the cytoskeletonlike triton shell of M. pneumoniae and localized primarily in the filamentous extensions of the mycoplasma cells.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

Quantitative aspects of immunogold labeling in embedded and in nonembedded sections

We tried to control immunolabeling conditions so that information about antigen concentration could be achieved by quantifying labeling patterns. Working with immunogold labeling procedures in ultrathin cryosections, we observed that differential penetration of immunoreagents causes considerable differences in labeling efficiency between various cell structures. Therefore, in these nonembedded sections, labeling densities can only be used to measure variations in antigen concentration within one cell structure. After embedding the tissue in 30% polyacrylamide (PAA), differences in penetration were negated. The equalizing effect of PAA on the labeling efficiency enabled us to design a simple immunocytochemical method by which concentrations of a protein can be measured in situ at subcellular levels, provided that no variations in the protein's structural conformation occur that would affect its immunoreactivity. In spite of a higher sensitivity observed for Ig-gold, we preferred to use protein A-gold in our system because of the low nonspecific labeling and the more precise antigen detection by the latter immunomarker.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

Quantitative immunogold localization of protein phosphatase 2B (calcineurin) in Paramecium cells.

For immunogold EM labeling analysis, we fixed Paramecium cells in 4% formaldehyde and 0.125% glutaraldehyde, followed by low-temperature embedding in unicryl and UV polymerization. We first quantified some obvious but thus far neglected side effects of section staining on immunogold labeling, using mono- or polyclonal antibodies (Abs) against defined secretory and cell surface components, followed by F(ab)(2)- or protein A-gold conjugates. Use of alkaline lead staining resulted in considerable rearrangement and loss of label unless sections were postfixed by glutaraldehyde after gold labeling. This artifact is specific for section staining with lead. It can be avoided by staining sections with aqueous uranyl acetate only to achieve high-resolution immunogold localization of a protein phosphatase on unicryl sections. In general, phosphatases are assumed to be closely, although loosely, associated with their targets. Because the occurrence of protein phosphatase 2B (calcineurin) in Paramecium has been previously established by biochemical and immunological work, as well as by molecular biology, we have used Abs against mammalian CaN or its subunits, CaN-A and CaN-B, for antigen mapping in these cells by quantitative immunogold labeling analysis. Using ABs against whole CaN, four structures are selectively labeled (with slightly decreasing intensity), i.e., infraciliary lattice (centrin-containing contractile cortical filament network), parasomal sacs (coated pits), and outlines of alveolar sacs (subplasmalemmal calcium stores, tightly attached to the cell membrane), as well as rims of chromatin-containing nuclear domains. In other subcellular regions, gold granules reached densities three to four times above background outside the cell but there was no selective enrichment, e.g., in cilia, ciliary basal bodies, cytosol, mitochondria, trichocysts (dense-core secretory organelles), and non-chromatin nuclear domains. Their labeling density was 4- to 8.5-fold (average 6.5-fold) less than that on selectively labeled structures. Labeling tendency was about the same with Abs against either subunit. Our findings may facilitate the examination of molecular targets contained in the selectively labeled structures. (J Histochem Cytochem 48:1269-1281, 2000)     

One antigen, one gold? A quantitative analysis of immunogold labeling of plasma membrane 5'-nucleotidase in frozen thin sections

We present an evaluation of the efficiency of immunogold labeling for a low abundance plasma membrane protein. Several independent methods were used to determine the density of 5'-nucleotidase on the plasma membrane of the Fao cell. These methods include morphometry in combination with either enzymology or cell surface radiometric assay. Immunocytochemistry of frozen thin sections with either single or double layers of antibody and visualized with protein A complexed with 5 nm colloidal gold was used to estimate the same density. The application of a balance sheet to immunogold labeling demonstrates that the labeling is never quantitative. For example, labeling of the cell surface is always greater than labeling on the section. We show that departures from the "one antigen, one gold" ideal are systematic, so that an efficiency can be calculated and quantitative results can be obtained. The ability to obtain reliable quantitative results from immunogold labeling extends the utility of this already powerful technique.     

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.     

Visualization of identified GFP-expressing cells by light and electron microscopy.

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1 microm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.     

Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.     

Quantitation in immunocytochemistry: correlation of immunogold labeling to absolute number of membrane antigens

Baby hamster kidney cells infected with Semliki Forest virus were used as a model system for quantitative immunocytochemical labeling studies. In this system, a well-characterized membrane protein complex is present in different concentrations in three separate locations. Using immunogold labeling of cryosections, we compared the number of gold particles labeling the membranes of endoplasmic reticulum, Golgi stack, and fully formed virions at the plasma membrane to the biochemically determined concentrations. The efficiency of labeling was 40, 13, and 14% for the three structures, respectively. In a comparative study, Lowicryl K4M sections were found to give significantly lower levels of labeling.     

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.     

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

Summary We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.     

Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.

A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and various non-isotopic nucleotide analogues. The labeled nucleotides bound to the surface of ultra-thin sections were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern was strongly dependent on the divalent cation used in the TdT medium. The method revealed with great precision the specific DNA-containing structures within Ehrlich tumor cells, even where DNA was present in very low amounts. The method is compatible with all usual fixation and embedding procedures and can be combined with cytochemical methods. The in situ TdT method provides a very useful tool for pinpointing the precise location of DNA within biological material at the ultrastructural level.     

Optimization of gold immunolabeling techniques on ultrathin slides obtained from samples fixed with glutaraldehyde and osmium tetroxide and enclosed in epoxy resin]

An immunogold labeling technique was carried out on plants infected with CMV, fixed with glutaraldehyde and osmium tetroxide and embedded in araldite CY 212. The effect of the type of support-film used, the resin and manipulations of the grids during immunogold steps, were studied and are discussed. The antigenic activity of virus was restored by treating the sections with sodium metaperiodate. The very high non-specific reactions observed with the support-film or with the resin were eliminated by adding powdered skimmed milk or non-purified albumin into the buffers. Purified bovine serum albumin (grade V) or chicken albumin (grade III to V) were inefficient in reducing this non-specific background.     

On-grid immunogold labeling of glial intermediate filaments in epoxy-embedded tissue

On-grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold-labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy-embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde-fixed, epoxy-embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on-grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween-20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy-embedded material to immunolabel thin structures like intermediate filaments.     

Immunoelectron microscopic localization of chlamydial lipopolysaccharide (LPS) in McCoy cells inoculated with Chlamydia trachomatis.

Interactions between Chlamydia trachomatis, host cells, and the immune system are believed to involve lipopolysaccharide (LPS). We used immunogold techniques to study the distribution of chlamydial LPS in cultured cells infected with C. trachomatis LGV-L1. McCoy cells inoculated with C. trachomatis were cultured and then fixed and embedded in situ with acrylic resins. Sections were immunolabeled with a protein A-gold method using antisera to the genus-specific, periodate-sensitive epitope on chlamydial LPS. Pre-embedding immunogold labeling on permeabilized cells was also done. By post-embedding methods, labeling for LPS was equally abundant over the outer membranes of elementary (EB) and reticulate bodies (RB). By post-embedding labeling, the sub-surface side of the EB outer membrane was more heavily labeled than the surface side. By pre-embedding labeling, LPS was found to be less abundant on the surface of EBs than RBs. Labeling for LPS was found over apparent lysosomes in McCoy cells and over electron-dense blebs on or near the surface of the plasma membranes of McCoy cells. These results indicate that the concentration of LPS in chlamydial membranes is constant during development but that with development its location changes from being mostly cell-surface to sub-surface. These results show that the post-embedding immunogold technique can be a useful approach for the cell culture-based study of chlamydial LPS.     

In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.     

An acetylcholinesterase method for in toto staining of peripheral nerves.

Stomach, small intestine, uterus, urinary bladder, vagina, mesentery, mesometrium and joint capsule of rats, gall bladder, cystic duct and bile duct of dogs and uteri of young children are stained in toto. Procedure: Tissue is perfused with saline containing hyaluronidase, then pinned on a flat layer of Paraplast and fixed for 24 hr in cold sucrose formol solution. Stomach, urinary bladder and gall bladder are also fixed in toto. Rinse for 2 days in cold 0.22 M sucrose in a sodium cacodylate buffer pH 7.2. Incubate in medium consisting of 60 mM acetate-buffer pH 5.0 or pH 5.6 (for human material only), 2 mM acetylthiocholine iodide, 15 mM Na citrate, 3 mM Cu sulphate, 0.5 mM K3Fe(CN)6, 5 times 10-4 M iso-OMPA, 1% Triton X 100 at 37C. Rinse in doubly distilled water. Dehydrate in glycerine/water mixtures of increasing glycerine content. Store in glycerine or delaminate under dissecting microscope. Delaminated specimens are mounted on gelatinized object glasses, cleared in xylene and coverslipped with Malinol. Specimens stored in glycerine can be studied microscopically. Stained specimens can also be embedded in Paraplast and sections can be studied after counterstaining.