Factor analysis of confocal image sequences of human papillomavirus DNA revealed with fast red in cervical tissue sections stained with TOTO-iodide

OBJECTIVE: To visualize and localize specific DNA sequences by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus (HPV) DNA was identified in cervical tissue sections with biotinylated DNA probes recognizing the whole genome of HPV DNA types 18 and 16, and DNA-DNA hybrids were revealed by streptavidin-alkaline phosphatase and Fast Red (FR). Cell nuclei were counterstained with TOTO-iodide. Image sequences were obtained using successive dynamic or spectral sequences of images on different optical slices from CLSM. The location of fluorescent signals inside tissue preparations was determined by FAMIS and/or selection of filters at emission. Image sequences were summarized into a reduced number of images, called "factor images," and curves, called "factors." Factors estimate spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between FR and nucleus staining in HPV DNA hybridization signals by taking into account differences in their spectral patterns and improved visualization by taking into account differences in their focus (depth emission profiles). CONCLUSION: FAMIS, together with CLSM, made possible the detection and characterization of HPV DNA sequences in cells of cervical tissue sections.     

Laser scanning confocal microscopy and quantitative microscopy with a charge coupled device camera improve detection of human papillomavirus DNA revealed by fluorescence in situ hybridization

Epithelial cervical CaSki, SiHa and HeLa cells containing respectively 600 copies of human papillomavirus (HPV) DNA type 16, 1–2 copies of HPV DNA type 16 and 10–50 copies of HPV DNA type 18 were used as model to detect different quantities of integrated HPV genome. The HPV DNA was identified on cell deposits with specific biotinylated DNA probes either by enzymatic in situ hybridization (EISH) or fluorescence in situ hybridization (FISH) involving successively a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and streptavidin-alkaline phosphatase complex or streptavidin-fluorescein isothiocyanate complex. With brightfield microscopy and EISH, hybridization spots were observed in CaSki and HeLa cells but hardly any in SiHa cells. With fluorescence microscopy and FISH, hybridization spots were clearly seen only on CaSki cell nuclei. In an attempt to improve the detection of low quantities of HPV DNA signals revealed by FISH, laser scanning confocal microscopy (LSCM) and quantitative microscopy with an intensified charge coupled device (CCD) camera were used. With both LSCM and quantitative microscopy, as few as 1–2 copies of HPV DNA were detected and found to be confined to cell nuclei counterstained with propidium iodide. Under Nomarski phase contrast, a good preservation of the cell structure was observed. With quantitative microscopy, differences in the number, size, total area and integrated fluorescence intensity of hybridization spots per nucleus were revealed between CaSki, SiHa and HeLa cells. Considered altogether our results shows that in situ hybridization is a powerful technique to detect small amounts of nucleic acid sequences but the choice of the technique for cell examination is important. Single genes of HPV were visualized most efficiently by association of FISH with LSCM or quantitative microscopy with an intensified CCD camera.     

The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction

Summary The parameter Tm^t has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tm^t) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tm^t is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tm^t is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.     

A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction.

We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.     

Detection of human papillomavirus in cervical scrapings by in situ hybridization and the polymerase chain reaction in relation to cytology

Summary A gynaecological out-patient population consisting of 200 patients aged 19–43 years (mean age 34.2 years) was screened for the presence of human papillomavirus (HPV) by the polymerase chain reaction and in situ hybridization on cervical scrapings. A novel method was applied for the detection of HPV in cervical cells by embedding them in a paraffin block before in situ hybridization was performed. This technique resulted in well preserved cytological morphology, easy performance and economy of probes. In eight of the 200 patients (4%), human papillomavirus DNA was revealed by the polymerase chain reaction. Subtyping revealed the presence of HPV serotype 16 DNA in three of these patients. In one patient HPV serotype 18 DNA was also present. The in situ hybridization assay was able to detect all those cases with a specific HPV serotype infection.     

Confocal microscopical analysis of epithelial cell heterogeneity in mouse Peyer's patches

Summary Cyanine dye fluorescence and alkaline phosphatase activities have been compared directly by confocal microscopy in a wide variety of cells present in the follice-associated epithelium of the mouse Peyer's patch to test the hypothesis that antigen-transporting M cells have a low membrane potential. In order to make these comparisons it was first necessary to equilibrate living tissue with the membrane potential sensitive dye DIOC₅(3), fix with glutaraldehyde and then incubate the fixed tissue with naphthol AS-BI phosphate, a substrate which is hydrolysed by alkaline phosphatase present in the luminal membrane of these epithelial cells. Naphthol AS-BI produced by this reaction, is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. Selecting the 488 nm wavelength of the argon laser source then allows one to measure alkaline phosphatase activities as Fast Red absorbance and membrane potentials by DIOC₅(3) fluorescence.Results obtained show a linear correlation between membrane potential and alkaline phosphatase activity. Relative lack of alkaline phosphatase activity, determined in fixed tissue, has been used previously to identify antigen-transporting M cells (Smithet al., 1987). The present work shows that it is now possible to recognize these cells in living tissue by measurement of DIOC₅(3) fluorescence. The possible importance of this finding in providing a way to study cell surface-antigen interactions taking place in living tissue is discussed.     

In situ hybridization: Alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections

Summary Alkaline phosphatase immunochemical systems were evaluated for use in the demonstration ofin situ hybridized biotin-labelled probes in frozen and fixed sections of tonsil. Three probes were used: total genomic DNA, pHY2.1, a human repetitive sequence which hybridizes to a 2.12 KB sequence on the Y chromosome (2000 repeats) and a 2.0 KB sequence on the autosomes (100–200 repeats), and human papilloma virus type II. Indirect, three- and five-stage detection methods were compared on cryostat sections. The indirect method involved the application of a streptavidin, biotinylated alkaline phosphatase sequence. The three-stage procedure comprised a mouse monoclonal anti-biotin, rabbit anti-(mouse immunoglobulin), mouse APAAP system. In the five-stage method the indirect and three-stage reagents were sequentially applied. Alkaline phosphatase was demonstrated using a Fast Red naphthol-capture method.The total genomic DNA probe was used initially to investigate hybridization conditions including the optimum temperature of denaturation, which was found to be higher than previously reported. The five-stage detection method gave the most sensitive results for the Y sequence probe, with intense demonstration of the Y body in male nuclei and autosomal sequences in female nuclei. This method was then applied to fixed tissue sections and gave Y body signals on Bouin's and Carnoy's fixed tissue. On the other hand tissue fixed using formalin-based solutions required proteolytic digestion as a pretreatment to hybridization for a Y body signal. The application of this methodology to viral diagnosis in routine fixed anogenital tissue and cytological preparations was also demonstrated.     

Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana

A procedure for whole mount fluorescence in situ hybridization (FISH) on plant tissue is reported. The technique was demonstrated on seedlings and flowers of Arabidopsis thaliana L. with rDNA as a probe, labelled, both for direct and indirect detection. It was found that fixation in 1% formaldehyde yielded the best results with respect to morphology and hybridization efficiency. The combination of whole mount FISH and confocal scanning laser microscopy allowed the nuclear localization of the rDNA loci in all tissues of both seedlings and flowers. Direct labelling yielded the best signal-to-noise ratio, especially in the apical zones of the seedlings. The technique was further illustrated on seedlings of A. thaliana in double labelling experiments with rDNA and a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei in all tissues in the seedling exhibited both signals, hybridization efficiency for both signals was reduced in the dense, apical zones as compared with single labelling experiments with rDNA.     

Spatial analysis of intranuclear human repetitive DNA regions by in situ hybridization and digital fluorescence microscopy

Summary Non-isotopic (fluorescent) in situ hybridization has established itself as a useful technique for the localization of DNA sequences in both metaphase and interphase cells. The rapid development of digital fluorescence microscopy, especially confocal microscopy, has become a powerful aid for the evaluation of the hybridization results in cytogenetic and cell biological applications. In this review we will demonstrate the utility of these methodologies for the three-dimensional visualization and analysis of chromosome-specific (peri)centromeric repetitive DNA sequences within the intranuclear structure of human cells and cell lines.     

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

Detection of mRNA and hnRNA using a digoxigenin labelled cDNA probe byin situ hybridization on frozen tissue sections

Summary A full-length cDNA clone encoding the constant region of T cell receptor chain was labelled by random priming DNA with digoxigenin-dUTP. The probe was used to estimate the relative amount of the receptor chain mRNA byin situ hybridization on frozen sections from human thymus and lymph nodes. The hybridization was visualized in blue using an anti-digoxigenin antibody conjugated with alkaline phosphatase and a subsequent enzyme-catalysed colour reaction. The distributions of the signal in tissue sections were as expected. Moreover, labelled cells showed hybrids both in the cytoplasm and in the nucleus, and strongly and weakly stained cells were clearly distinguishable. The results indicate that this method ofin situ hybridization should be useful in the detection of specific mRNA in frozen sections.     

Study of stringency conditions for human papillomavirus DNA detection on cell lines,frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes

Summary In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Siha and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35° C below the melting temperature of DNA (Tm-35° C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35° C; C-medium stringency, hybridization at Tm-35° C and washings at Tm-12° C; D-high stringency, hybridization at Tm-12° C and washing without formamide; E-very high stringency, hybridization and washings at –12° C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes.When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type.Stringent (Tm-12° C) and non stringent (Tm-35° C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV. Nineteen samples were totally negative under both NSC and SC, and considered as non-infected by HPV. In 22 specimens positive, under both NSC and stringent conditions (SC), the HPV type was identified. Ten specimens reacted with 1, 2 or 3 HPV types under NSC but the HPV DNA was not typed with the probes used. Six lesions were negative under NSC but were typed under SC. Most paraffin sections were labeled only with one HPV probe under NSC, whereas frozen sections were often labeled with 2 or 3 HPV probes. The HPV probe positive under SC was usually positive under NSC in both frozen and paraffin sections. HPV type 1 probe was more frequently positive under NSC in paraffin- embedded sections than the others and the 4 probes tested were equally positive in frozen sections.These findings show the interest of in situ hybridization in low stringency conditions since 17% of our lesions (10/57) were positive only under NSC: HPV DNA was detected but not typed with the probes used. Frozen sections were more frequently positive than paraffin sections, suggesting a loss of DNA accessibility in the latter, due to the fixation or processing before hybridization.     

Improved detection of in situ hybridization by laser scanning confocal microscopy.

Laser scanning confocal microscopy (LSCM) offers a significant improvement over conventional bright-field and dark-field light microscopy for producing images of silver grains in autoradiograms of specimens prepared by in situ hybridization. The out-of-focus image of the background silver grains present in the emulsion is eliminated from the in-focus image of the radioactive probe associated with the cells by optical sectioning with the LSCM operated in a reflected light mode. The improved images produced by the LSCM provide a significant increase in the sensitivity of detecting positively labeled cells and tissues prepared by in situ hybridization. The power of this detection method is demonstrated using samples of HIV-infected human peripheral blood cells, tissue sections of human placenta and human skin. It is anticipated that the method can be universally applied to samples prepared by in situ hybridization techniques.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Summary Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV 16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF-or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidinbiotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV 16 DNA and haptenized HSV2 DNA.     

Combination of lamin immunocytochemistry and in situ hybridization for the analysis of chromosome copy numbers in tumor cell areas with high nuclear density

We describe the application of lamin immunocytochemistry (ICC) and single- or double-target fluorescence in situ hybridization (FISH) on 4 microm thick frozen tissue sections as a method to facilitate scoring of aberrant chromosome copy numbers in colonic tumors. Analysis of FISH signals in colon tissue sections is often hampered by overlap and truncation of epithelial nuclei, due to the density of the epithelial cells. Furthermore, on the basis of nuclear staining it is often difficult to determine whether or not nuclei are overlapping, or adjoining. Therefore, reliable evaluation of (F)ISH signals to screen for genomic changes was until now mainly restricted to isolated nuclei obtained from relatively thick tissue sections. In this study the applicability of lamin ICC, to stain the nuclear periphery and to distinguish individual nuclei, combined with the FISH procedure is explored to solve this problem for colon epithelium. For ICC we applied the alkaline phosphatase (APase)-Fast Red detection method, since the fluorescent precipitate of this reaction resists extensive proteolytic digestion as needed for efficient FISH on tissue sections. Chromosome copy numbers could easily be determined in 4 microm thick frozen tissue sections by combining lamin ICC and FISH. The ratio of the copy numbers of the chromosomes 7 and 17 could be determined in frozen tissue sections after combined lamin ICC and double-target FISH. It is concluded that the combination of lamin ICC and FISH improves chromosome copy number analysis and can be used to investigate genomic changes in different tumor compartments in thin frozen tissue sections.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF- or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidin-biotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV16 DNA and haptenized HSV2 DNA.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.     

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

A one-day double-labelling technique for tissue specimens: Immunogold-silver staining forin situ hybridization combined with alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for antigens

Summary An improved technique is described that addresses the problems of sensitivity, specificity, the use of hazardous radioactive equipment and time consumption in immunohistochemical labelling and double labelling ofin situ hybridization of tissue specimens. It consists of a two-step protocol in which digoxigenin-uridine triphosphate (UTP) labelled riboprobes in thein situ hybridization step are visualized by the immunogold-silver staining method, and double labelling of tissue antigens is achieved by the application of an alkaline phosphatase-anti-alkaline phosphatase staining step. We tested this protocol using snap-frozen tissue sections of synovial tissue from patients with rheumatoid arthritis. The target mRNA was detected by perforin or cathepsin D riboprobes, the double labelling was performed using anti-collagen type IV and alpha-smooth muscle actin antibodies. It is concluded that, in comparison with an established three-to four-day double-labelling protocol used in many laboratories, this one-day combination is currently the most rapid assay of reliable quality for double labelling ofin situ hybridization products and tissue antigens.