Methanol Fixation Research Articles

Preservation of comet assay slides: comparison with fresh slides

The single cell gel electrophoresis assay (comet assay) is an inexpensive, rapid and highly sensitive method for the determination of DNA damage, crosslinks, and alkaline-labile lesions in individual cells. A limitation of the procedure is that the microelectrophoretic gels must be scored rapidly as the comet configuration deteriorates on storage due to dehydration of the agarose and diffusion of DNA. The objectives of this study were firstly to evaluate drying regimes as rapid and simple methods of preservation of the microgels as close to their original fresh state as possible, and secondly to examine the effects of storage of the slides. Human hepatoma (HepG2) cells challenged for 30 min with hydrogen peroxide (H 2O 2) were used in the study. Microgel slides were prepared and evaluated immediately, or after drying with or without a methanol fixation step. Microgels that were dried at a variety of temperatures (22–50°C) and re-hydrated did not differ in the values obtained for H 2O 2-induced DNA damage when compared to fresh samples. Samples could also be continually dried and re-hydrated over a period of up to 3 months with no obvious loss of information. In conclusion, drying of microgels represents a simple and inexpensive method of preserving comet assay slides.

Reduced invasion of cultured cells pretreated with a monoclonal antibody elicited against refractile body antigens of avian coccidial sporozoites.

The effect of a monoclonal antibody (1209-C2) elicited against sporozoite refractile-body antigen on invasion of cultured baby hamster kidney cells by avian Eimeria species was examined in vitro. Pretreatment of sporozoites with 1209-C2 for 45 min before inoculation into cultures or simultaneous introduction of sporozoites and 1209-C2 into cultures had no significant effect on invasion. However, pretreatment of cultures for 45 min with 1209-C2 (also with media from other cloned 1209 cell lines) significantly inhibited invasion by sporozoites of Eimeria tenella and E. adenoeides. Pretreatment of cultures with 2 unrelated monoclonal antibodies with the same isotype as 1209-C2 did not inhibit invasion by E. tenella. There was a significant correlation between time of exposure of the cultures to 1209-C2 and invasion (r = -0.80924; p = 0.0001), with inhibition of invasion occurring after 20 min exposure, but not after 10 min. There was also a significant correlation between the titer of 1209-C2 and invasion (r = 0.62291; p = 0.0305). Monoclonal antibody 1209-C2 cross-reacted with epitopes of baby hamster kidney cells by both immunofluorescence and Western blot. The fluorescent labeling of the cells differed according to the fixative that was used. In formalin-fixed cultures labeled with 1209-C2, fluorescent foci were distributed over the entire cell; after methanol fixation, 1209-C2 reacted with only discrete foci in the nucleus. On Western blots of sporozoites, 1209-C2 reacted with antigens having molecular sizes of approximately 8, 17, 23, 30, and 45-60 kDa, and with several minor bands. On baby hamster kidney cells, the antibody reacted primarily with bands of approximately 30, 45-60, and slightly with other bands. The data suggest that interactions among similar molecules in the sporozoites and host cells may play a role in cellular invasion.

Expression of COX-2 by Normal and Reactive Astrocytes in the Adult Rat Central Nervous System

We have used a previously characterized antiserum against cycloxygenase-2 (COX-2) together with cold methanol fixation to immunohistochemically locate the protein in astrocytes in rat brain. Although in cerebral cortex most enzyme was located in neuronal perikarya as previously described, a number of glial fibrillary acidic protein (GFAP)-positive astrocytes were also labeled. No COX-2-positive neurons were seen in the cerebellum, but here also a subset of GFAP+ astrocytes was present which contained the enzyme. The number of COX-2-positive astrocytes increased considerably after injection of the neurotoxin kainate into the cerebellum. These immunohistochemical data were supported by semiquantitative RT-PCR results, which were used to assess the levels of COX-2 mRNA relative to the housekeeping gene hypoxanthine phosphoribosyl transferase. PGE2levels were measured in contralateral and lesioned cerebellum to correlate changes in COX-2 immunoreactivity and mRNA with physiological events. PGE2levels increased by 230% in the lesioned cerebellar hemispheres in comparison to the contralateral ones. We discuss the possibility that the targets for astrocytic prostaglandins might include both autocrine effects and paracrine responses of neurons, lymphocytes and capillary endothelial cells.

A convenient method of DNA extraction from blood anticoagulated with EDTA.

Surplus blood often remains after routine clinical tests using EDTA-anticoagulated samples. To use this blood for DNA analysis, we isolated white cells by adherence to polyethylene terephthalate fibers, which could be stored for several weeks transported by mail after methanol fixation. DNA yield was sufficient and correlated with white cell count. Extracted DNA was free of hemoglobin contamination and durable to polymerase chain reaction and enzyme digestion, which yielded products visualized as well-separated bands on electrophoresis. We found our method to be practical for the routine clinical laboratory.

Quantitative assessment of human sperm acrosome reaction by using fluorescein isothiocyanate conjugated concanavalin A--comparison between highly purified acrosome reacted with non-acrosome reacted sperm.

In the present study, to achieve quantitative assessment of human sperm acrosome reaction by using fluorescein isothiocyanate conjugated Concanavalin A (FITC-Con A), the optimum conditions for microscopic observation were established by comparing the fluorescent images between the purified human sperm with the intact acrosome (non-acrosome reacted sperm; non-AR sperm) and those with exposed inner acrosomal membrane (acrosome reacted sperm; AR sperm). FITC-Con A stained not only the inner acrosomal membrane but also the other portions. These non-specific bindings were excluded by the competitive dissociation with alpha-D-mannose (Man) solution, the specific labeling of the inner acrosomal membrane with FITC-Con A was accomplished in the presence of 0.5 mg/mL Man. The influence of methanol fixation on the acrosome status were examined. The assessment of the acrosome reaction should evaluate whether the inner acrosomal membrane is exposed or masked by the plasma membrane, and methanol fixation is often employed in the histochemical stain. By methanol fixation, the fluorescent profiles of AR sperm were not altered before and after the treatment, bright fluorescence were found in the acrosomal regions. On the other hand, non-AR sperm gave weak fluorescence on the sperm heads before the treatment, the acrosomal regions fluoresced brightly after the treatment similar to those of AR sperm. Methanol fixation caused false positive results, it might denaturate the plasma membranes and facilitate permeability of FITC-Con A. FITC-Con A fluorescent stain and the modified triple stain (rose bengal stain) gave good positive correlation to evaluate AR sperm. This result concluded that the optimum conditions with FITC-Con A stain in the present procedure might be an useful tool for observation of human sperm acrosome reaction.

Enzymatic isolation and culture of cement secreting cells from cypris larvae of the barnacle Megabalanus rosa

Isolation of viable secretory cells from cyprid cement glands of the barnacle Megabalanus rosa was achieved by trypsin digestion followed by mechanical treatment. Isolated cells, which were classified into two groups based on cell and granule size, were comparable to earlier descriptions of α‐ and ß‐cells in the cement glands of Balanus balanoides. "α‐Cells”; were large, elongated and contained many small secretory granules (α‐granules), while “ß cells”; were small and contained several larger granules (ß‐granules). α‐Granules expanded significantly when exposed to solutions containing low NaCI concentrations after methanol fixation, whilst ß‐granules did not, suggesting differences in the properties of the respective granules. Isolated cells were cultured for up to one week. The potential application of in vitro cell culture for physiological studies is presented.

Modified formalin and methanol fixation methods for molecular biological and morphological analyses.

Several simplified fixation methods were examined to determine their suitability for both molecular biological analyses and morphological study. Fixation with 10% v/v formalin alone at 4 degrees C and containing 5 mmol/L ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) at room temperature preserved significantly more high-molecular-weight DNA than 10% v/v formalin fixation at room temperature. The morphological differences between tissues fixed using these modified formalin fixation methods and conventional 10% v/v formalin fixation were negligible. Of the dehydration fixatives tested, 100% methanol did not cause regional differences due to artificial tissue shrinkage and the morphology of sections prepared by methanol fixation was preserved consistently better than that of acetone- or ethanol-fixed sections. All three dehydration fixatives preserved relatively higher-molecular-weight DNA and RNA, compared with formalin. Cold formalin, formalin containing EDTA at room temperature and 100% methanol are recommended as standard and additional fixatives routine clinicopathological laboratory use.

Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides.

Understanding the distribution of tumor-associated antigens on cancers and normal tissues is essential for selection of targets for cancer immunotherapy. Seven carbohydrate antigens, potential targets for immunotherapy, were studied using a panel of well-characterized MAbs by immunohistochemistry on cryostat-cut tissue sections of 13 types of cancers and 18 normal tissues. GD2 and GD3 were present on most cancers of neuroectodermal origin and GD2 was also present on B cell lymphomas. 9-O-acetyl-GD3 was detected only on melanoma while fucosyl GM1 was detected only on small cell lung cancers (SCLC). Surprisingly, GM2 was strongly expressed on all tested tumors, including cancers of neuroectodermal origin and cancers of epithelial origin. Polysialic acid was primarily expressed on SCLC and neuroblastomas. Globo H was present on most cancers of epithelial origin. These antigens were also identified in normal tissues. Fucosyl GM1 was not expressed significantly on any of the normal tissues analyzed. GD3, GD2, GM2 and polysialic acid were detected in normal brain to varying degrees. GM2 and Globo H were expressed on the luminal surface of epithelia of a variety of organs. The unexpected expression of GM2 on a broad range of cancers and normal epithelial tissues was confirmed by loss after methanol fixation and by immune thin layer chromatography.

Essential role for gamma -tubulin in the acentriolar female meiotic spindle of Drosophila

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin.

A fast comet assay variant for solid tissue cells. The assessment of DNA damage in higher plants

The single-cell gel electrophoresis or comet assay, under high alkaline conditions, detects low levels of DNA damage. In it, broken DNA migrates from the nucleus to the anode providing images similar to comets. To adapt this assay to solid tissue cells, nuclei were directly obtained from Allium cepa L. roots. The surface of each single fresh sharply cut meristem was exposed to a small drop of 50 mM Sörensen buffer at pH 6.8, placed on a regular agarose-coated slide. By immediately adding low melting point agarose at 30°C, nuclei resulted embedded in agarose. A final layer of this agarose ended the preparative steps. Conventionaly prepared leukocytes were used as a control. The treatment with detergent (lysis step of the conventional assay) proved to be unnecessary for the nude nuclei. A 20 min-long electrophoresis (at 0.65 V·cm −1, 230 mA and 10°C) was more sensitive than a 10 min-long one for detecting the differential response of plant nuclei to 2 and 4 Gy of γ-irradiation. A short fixation in methanol transformed the preparations into semi-permanent ones, without altering their later DNA staining by ethidium bromide. The use of instantaneously isolated nuclei simplifies and expands the use of this technique to any eukaryotic cell from solid tissues.

TGN38-green fluorescent protein hybrid proteins expressed in stably transfected eukaryotic cells provide a tool for the real-time, in vivo study of membrane traffic pathways and suggest a possible role for ratTGN38.

The green fluorescent protein (GFP) of Aquorea victoria is fluorescent when expressed as a recombinant protein in eukaryotic cells and has been used as a convenient marker of gene expression in vivo. It has also been used as a marker of the intracellular targeting of recombinant fusion proteins (part GFP, part protein of interest) which have been transiently expressed in eukaryotic cells grown in tissue culture. Thus, the use of GFP has proved a useful tool to study intracellular events in real-time. However, some transiently transfected cells fail to express, or localise correctly, the GFP-tagged protein. Therefore the production of stable cell lines expressing GFP-tagged integral membrane proteins may be essential for long-term studies. The generation of stably transfected eukaryotic cells expressing an integral membrane protein with a known, but poorly characterised intracellular trafficking pathway, would provide useful reagents for future, more precise, analysis of that pathway. TGN38 is a type I integral membrane protein which cycles between the trans-Golgi network (TGN) and cell surface; at steady state it is localised to the TGN. As such, TGN38 is an ideal candidate for tagging with GFP. We have generated cDNA constructs encoding ratTGN38 tagged at either the N- or C terminus with GFP. Transiently transfected rat (NRK) cells expressed active fluorophore, but failed to show correct localisation of the fusion protein. In contrast, both constructs are appropriately localised in stably transfected NRK cells and both are fluorescent. Furthermore, the recombinant GFP-tagged proteins and the endogenous TGN38 molecules show identical responses to drugs and temperature blocks known to perturb intracellular morphology and membrane traffic pathways. In fact morphological changes to the TGN induced by brefeldin A were observed at earlier time points than had been described previously using immunofluorescence analysis of fixed cells, thus validating the use of in vivo, real-time analysis of GFP-tagged proteins. In addition, we show that (in contrast to the situation in COS cells) elevated expression of ratTGN38 in NRK cells does not lead to a fragmentation of the TGN; this has implications for the role which TGN38 is playing in the maintenance of the morphology of the TGN. The data we present demonstrate that: (i) it is possible to generate stable cell lines expressing integral membrane proteins tagged with GFP; (ii) the GFP tag remains fluorescent when expressed on either the cytosolic or the lumenal side of all membranes of the secretory pathway up to and including that of the TGN; (iii) the GFP tag does not interfere with the transport of TGN38 along the secretory pathway or its retention in the TGN; (iv) GFP remains fluorescent in cells which have been processed for immunofluorescence analysis (using either paraformaldehyde or methanol fixation); and (v) TGN38 plays a role in maintaining the morphology of the TGN. Thus, stably transfected cells expressing GFP-tagged integral membrane proteins can be used as effective tools for the real-time study of intracellular morphology and membrane traffic pathways in eukaryotic cells.

Effect of XPA gene mutations on UV-induced immunostaining of PCNA in fibroblasts from xeroderma pigmentosum group A patients.

We have investigated the relationship between XPA gene mutations and PCNA complex formation in the nucleotide excision repair (NER) process utilizing cells derived from various xeroderma pigmentosum group A (XP-A) patients. The PCNA complex formation was detected by PCNA immunostaining following methanol fixation. Results indicated that UV-induced PCNA staining at early stages was well correlated to the function of XPA protein and provided evidence that XPA protein-related recognition step was tightly linked to PCNA-associated events in the NER process in vivo.

Immunohistological demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies

Feline infectious peritonitis (FIP) virus antigen was demonstrated after methanol, ethanol or formalin fixation in paraffin-embedded tissues by means of monoclonal and polyclonal antibodies. The monoclonal antibody was induced by immunization with transmissible gastroenteritis virus. Polyclonal antibodies were obtained by purification on protein A-Sepharose of ascites fluid from a cat with FIP. Almost all cats diagnosed as suffering from FIP by postmortem and histological examination exhibited FIP virus (FIPV) antigen in macrophages in granulomas whereas FIPV antigen was only once demonstrable in another location.

A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures

Protamine crosslinking by disulphide (-SS-) bonds in the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine-rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatic. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smear from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet-blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro- and macro-spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho-/metachromatic ration seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in -SS- crosslinking.

Methodologies for the preservation of proliferation associated antigens PCNA, p120, and p105 in tumor cell lines for use in flow cytometry

The retention of antigen expression of PCNA, p120, and p105 in two tumor cell lines (MOLT-4 and MDA-MD-175-VII) under various conditions of fixation was investigated using flow cytometric analysis. Four currently utilized procedures for fixation/permeabilization of intracellular antigens were compared for their ability to stain the nuclear antigens. A procedure using a brief incubation in a solution of lysolecithin in paraformaldehyde followed by fixation in ice-cold methanol prior to antibody staining was selected to evaluate reagent protocols aimed at preserving antigen expression. Holding samples overnight at 4 degrees C in 2.5% fetal bovine serum after the lysolecithin/paraformaldehyde and methanol fixation steps prior to staining with monoclonal antibodies resulted in no decline in the percentage of cells positively stained for all three markers with little decrease in intensity of fluorescence and no increase in DNA coefficient of variation (c.v.). Fixed/permeabilized MOLT-4 cells held longer than 24 h before staining were lower in PCNA fluorescence than freshly stained cells; holding samples of either cell line longer than 48 h resulted in decreased PCNA and p120 staining. Prefixing and holding cells in 50% methanol or 50% ethanol overnight before processing and staining severely depressed PCNA and p120 fluorescence. Prefixing either cell line in a range of concentrations (0.25-1.0%) of paraformaldehyde also resulted in reduced intensity of PCNA and p120 fluorescence along with increased DNA c.v. P105 staining appeared to be relatively unaffected by all prefixation/storage conditions tested, except for a decline of fluorescence when MDA cells were prefixed in 50% ethanol. Cells cryopreserved in liquid nitrogen for 1 week before processing showed < 5% loss of PCNA and p120 fluorescence compared to freshly processed cells, but p105 fluorescence dropped 29% in cryopreserved MDA cells. These results underscore the fact that specific protocols for the fixation and storage of biological samples prior to staining and analysis must be determined for the specific nuclear antigen marker under investigation.

Lipopolysaccharide in cells infected by Chlamydia trachomatis.

In view of the controversy concerning the expression of chlamydial lipopolysaccharide (LPS) in the eukaryotic host cell, the presence of this molecule was examined in three cell types which were experimentally infected with Chlamydia trachomatis serotype E. LPS was detected in the McCoy cell line, human endometrial epithelium and human amniotic epithelium with two monoclonal antibodies. The appearance and distribution of LPS at the host cell surface during the chlamydial developmental cycle and its transmission to neighbouring cells were examined by immunofluorescence microscopy after air drying of the host cells. LPS distribution was not uniform; it was first observed on regions of the cell surface in close proximity to the chlamydial endosome (inclusion). Soon after, the antigen was also detected at points of contact with neighbouring uninfected cells. Immunofluorescent plaques of host cells contaminated with LPS were thus formed in the vicinity of infected cells. These plaques increased in size over 2 d before becoming smaller as host cell lysis occurred. The major outer-membrane protein (MOMP) was not visualized on the host cell surface after air drying. No cell-surface LPS antigen was observed in live cells or those fixed in formaldehyde without air drying. Conventional methanol fixation and immunolocalization of LPS and MOMP in parallel infected cultures stained these antigens within inclusions in the expected fashion. Radio-immunoassays were used to quantify LPS in confluent McCoy cell monolayers during the chlamydial developmental cycle. Cell-surface-associated and inclusion-associated LPS, measured by direct binding of 125I-labelled anti-LPS monoclonal antibodies to air-dried or methanol-fixed monolayers respectively, increased for up to 3 d then declined.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of mouse sperm using monoclonal anti‐sperm antibody A‐1

We have previously prepared an anti-mouse sperm monoclonal antibody (A-1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A-1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol-fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A-1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence-positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A-1 antibody was reduced to 55% by incubation in zona-containing media from the 80% obtained in zona-free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A-1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative flow cytometric analysis of proliferating cell nuclear antigen (PCNA) antibodies in human solid neoplasms

Proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase delta, associated with DNA synthesis, is increasingly used to determine tumor growth rate. To determine the growth fraction by PCNA, various antibodies and fixation procedures are being used. We assessed the flow-cytometric measurement of PCNA using three monoclonal antibodies (PC10, 19F4, and 19A2) and two fixation protocols (paraformaldehyde and methanol) in tissue-culture cell lines and human solid neoplasms to determine their potential clinical application. Thirty-one solid tumors and four normal tissues were analyzed, along with MOLT-4 and HL-60 cell lines as positive controls and normal peripheral blood lymphocytes as a negative control. PC10 with methanol fixation consistently detected higher PCNA positivity in human solid neoplasms than 19F4 and 19A2. Using PC10, the differences in positivity among fixation methods occurred in the G0/1 phase, not in S + G2M, of the cell cycle. No correlation was found between PCNA positivity and tumor grade and DNA ploidy in tumors analyzed. A statistical correlation was found between overall PCNA positivity and RNA content as determined by acridine orange analysis. The growth fraction by PCNA in solid neoplasms was most reliably determined by PC10 with methanol fixation.

Effects of selected chemotherapeutic agents on PCNA expression in prostate carcinoma cell lines.

Bivariate flow cytometric analysis of proliferating cell nuclear antigen (PCNA) was performed on prostate carcinoma cell lines (PC-3, DU-145). For both cell lines 100% methanol fixation provided optimal fluorescence intensity of PCNA. The ratio of PCNA/DNA increased in late G1 through early S/phase, followed by a decrease in mid- and late S and enhancement in G2/M phase. PCNA expression was increased in G2/M phase cells treated for 48 h with vinblastine. A slight decrease in PCNA expression was observed with cyclohexamide treatment. Hydroxyurea induced an increase in S-phase fraction along with enhanced PCNA expression. Methotrexate and Adriamycin had little effect on the cell cycle compartments of PC-3 or DU-145; however, methotrexate decreased PCNA expression, while Adriamycin enhanced it. Cisplatin increased S-phase in both cell lines, increasing PCNA expression in PC-3 and decreasing it in DU-145 cells. The data on the effects of drug treatment point to a dissociation between PCNA expression and S-phase fraction as calculated from the DNA distribution. In some cases, e.g., the cisplatin studies, different effects were obtained in the two different cell lines treated with the same drugs. Whether changes in PCNA expression will provide more useful information than S-phase fraction for evaluation of potential antitumor drugs is not known.

Expression of the major outer membrane protein of Chlamydia trachomatis in Escherichia coli

The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein.