Quantitative In Situ Hybridization Research Articles

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

Abstract 3626: Quantitative analysis of microRNAs by in situ hybridization reveals prognostic value of miRNAs in melanoma and breast cancer

Abstract Introduction: Most miRNA measurement methods require total RNA extraction which destroys spatial information and makes standardization a challenge. In situ hybridization (ISH) however, allows the direct assessment of miRNA expression levels in tissue and importantly allows for the evaluation of expression in malignant cells as well as stromal cells. We have developed and validated a novel method for the quantitative analysis of miRNA expression by ISH on formalin fixed paraffin embedded tissue microarrays (TMAs) using miR-21, miR-92a, miR-34a, miR-221, miR-205, and Let-7a. Methods: In order to quantitatively measure miRNA expression by ISH, DIG labeled LNA modified miRNA probes were employed and multiplexed with DAPI and cytokeratin or S100/GP100 immunofluorescence to apply the AQUA technology. This co-localization based, quantitative approach allows for the measurement of miRNA expression in subcellular compartments. The assay allows standardization of miRNA measurement and potential for rapid large cohort assessment. We also assessed protein targets of each miRNA by AQUA to investigate the hypothesized inverse relationship between miRNAs and target proteins. Results: In hundreds of tissue samples a broad dynamic range and a variable expression pattern specific to each miRNA was observed. Specificity of the assay was validated with anti-miR and miRNA mimic transfected cell lines, miR-21 knock out mouse tissue, and blocking oligo experiments. The assays were performed on near serial sections and were found to have high reproducibility with R*2>0.8. miR-221 and miR-205 were found to be prognostic in breast cancer and melanoma respectively (p<0.05). Finally, the inverse relationship between miRNAs and putative targets were not seen in the large population cohorts. Conclusions: This specific and reproducible method for the quantitative analysis of miRNA expression provides proof of concept for the use of miRNAs as tissue biomarkers using quantitative ISH. Future studies will further determine the prognostic value of miR-221 and miR-205, and the relationship between miRNAs and target protein expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3626. doi:1538-7445.AM2012-3626

Abstract LB-480: MicroRNA in situ hybridization techniques: Co-detection of target protein and image analysis-based quantitation

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that constitute a novel group of biomarkers with exciting functions in cell differentiation, proliferation and apoptosis by mediating degradation or destabilization of target mRNAs. Expression profiling provides identification of disease-related miRNAs; however, questions towards molecular interactions in vivo remain un-answered. In situ hybridization (ISH) allows visualization the miRNAs in histological sections and thereby allows determination of specific cellular origin, which is an essential step for understanding the miRNAs role in any biological setting. We have developed fast double fluorescence and quantitative ISH technologies that facilitates interpretation of roles for miRNA in cancer progression. On a semi-automated platform, HistoFlex, the double fluorescence assay can be completed within 2 hours without loss of sensitivity and specificity. In order to characterize the miR-21 positive cells in the breast cancer stroma, we combined miR-21 ISH with smooth muscle α-actin in a double fluorescence assay, and found that most, but not all miR-21 positive cells, are smooth muscle α-actin positive and include both myofibroblasts and myoepithelial cells in the neoplastic tissue. Subsequent combination of miR-21 ISH and programmed cell death-4 (PDCD4) immunofluorescence showed differential expression in many but not all miR-21 positive cells. The findings suggest that PDCD4 (mRNA), as a predicted miR-21 target according to sequence alignment, may also take place in vivo. Thus, a role of miR-21 in cell survival and apoptosis is likely. miRNA ISH also allows quantitative estimation of miRNA expression levels in archived formalin-fixed and paraffin embedded tissue samples. Using chromogenic detection of miRNA and subsequent image analysis, semi-quantitative expression estimates can be obtained, which can be used for correlation with clinical parameters, such as drug response, diagnosis, prognosis and other molecular profiles. We have established an automated 1-day assay that provides quantitative estimates with a precision of app. 15%. We also show that a quantitative estimate obtained in one paraffin section with tumor tissue is representative for the whole tumor. For clinical applications, we present data showing that the miRNA ISH signal can be quantified in routinely processed paraffin samples from colon and breast cancer, and that this approach can provide novel diagnostic possibilities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-480. doi:1538-7445.AM2012-LB-480

OPG and Rankl Expression in Osteoblasts Grown on Different HA Ceramics

Hydroxyapatite (HA) ceramics is one of the most widely used bone substitute in clinics. Limited information is available concerning how HA ceramics may affect osteoprotegerin (OPG) and RANKL expression. In this study, osteoblastic-like SaOS-2 cells were grown on HA ceramics sintered at different temperature for 3 and 6 days, RANKL and OPG mRNA expression were analyzed with quantitative in situ hybridization (QISH) technique. Result showed that SaOS-2 grown on HA ceramics sintered at 800°C expressed higher RANKL mRNA than on other two HA ceramics after 3 days’ culture. No significant difference in OPG expression on different surfaces was detected after 3 days and 6 days culture. This result suggests that HA sintered at low temperature tend to induce more bone remodeling after implantation.

Diverse patterns of cell-specific gene expression in response to glucocorticoid in the developing small intestine

Although glucocorticoids are known to elicit functional maturation of the gastrointestinal tract, the molecular mechanisms of glucocorticoid action on the developing intestine have not been fully elucidated. Our previous microarray studies identified 66 transcripts as being rapidly induced in the jejunum following dexamethasone (Dex) administration to suckling mice. Now we report the specific cellular location of a subset of these transcripts. Mouse pups at P8 received Dex or vehicle and intestinal segments were collected 3-4 h later. Robotic-based in situ hybridization (ISH) was performed with digoxygenin-labeled riboprobes. Transcripts studied included Ndrg1, Sgk1, Fos, and two unknown genes (Gene 9 and Gene 36). As predicted, ISH revealed marked diversity of cellular expression. In small intestinal segments, Sgk1 mRNA was in all epithelial cells; Fos mRNA was confined to epithelial cells at the villus tip; and Ndrg1 and Gene 36 mRNAs were localized to epithelial cells of the upper crypt and villus base. The remaining transcript (Gene 9) was induced modestly in villus stroma and strongly in the muscle layers. In the colon, Ndrg1, Sgk1, and Gene 36 were induced in all epithelial cells; Gene 9 was in muscle layers only; and Fos was not detectable. For jejunal segments, quantitation of ISH signals in tissue from Dex-treated and vehicle-treated mice demonstrated mRNA increases very similar to those measured by Northern blotting. We conclude that glucocorticoid action in the intestine reflects diverse molecular mechanisms operating in different cell types and that quantitative ISH is a valuable tool for studying hormone action in this tissue.

Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR

In situ hybridization (ISH) is an essential technique for mapping gene expression in the brain. Although many ISH protocols provide for quantitative analysis of individual mRNAs in different brain regions or across experimental conditions, this technique has lacked the necessary standardization for quantitative comparisons between different mRNA transcripts. We have developed a standardized quantitative ISH (SQuISH™) protocol that utilizes multiple radioactive oligonucleotide probes, providing for increased sensitivity, decreased background and accurate comparison of relative mRNA levels. We evaluated the SQuISH™ protocol against a riboprobe-based ISH procedure by comparing the mRNA expression levels in the brain for two transcripts, insulin receptor substrate p53 (IRSp53) and Calsenilin. The results of these two methods were then validated by real-time quantitative PCR. Both protocols exhibited identical mRNA expression patterns for IRSp53 and Calsenilin. In three brain regions analyzed, the levels of IRSp53 mRNA expression were ∼1.5-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure, although the relative abundance in regional expression levels was similar between the two methods. In contrast, the levels of Calsenilin mRNA expression were 10–17-fold higher with the riboprobe-based ISH than with the SQuISH™ procedure and the relative abundance in regional expression levels was different. When compared to the real-time PCR results, the SQuISH™ method showed almost identical relative levels of IRSp53 to Calsenilin mRNA in all three brain regions analyzed, while the riboprobe-based procedure showed a completely opposite trend. These results support the accuracy of the SQuISH™ protocol for determining relative mRNA levels in the brain.

Osmotic regulation of angiotensin AT1 receptor subtypes in mouse brain

Experiments were performed to study angiotensin (Ang) AT1a and AT1b mRNA expression in mice, including, examination of brain distribution and the effect of salt loading. In situ hybridization (ISH) methods showed that the pattern of mRNA expression was identical for AT1a and AT1b, with cellular labeling in rostral forebrain, hypothalamus and brainstem. Receptor mRNAs were concentrated in brain regions involved in the regulation of electrolyte and cardiovascular balance. Immunocytochemistry with AT1 specific antisera showed a pattern that was consistent with the ISH. Reverse transcriptase-polymerase chain reaction (RT-PCR) of hypothalamus and pituitary verified the presence of both AT1a and AT1b mRNA. Using quantitative ISH, we found that AT1a mRNA expression was significantly increased after 5 days of 2% NaCl consumption in anterior third ventricle (AV3V), paraventricular hypothalamus (PVN) and subfornical organ (SFO), but unchanged in anterior pituitary. There were no significant changes in AT1b mRNA. These results document the utility of ISH coupled with quantitative imaging techniques for the study of subtype specific expression. Using ISH and RT-PCR, we verified that AT1a and AT1b receptors are expressed in mouse brain and pituitary and show a similar pattern of distribution. Salt loading produced a specific increase in AT1a mRNA in osmosensitive regions, suggesting that this receptor subtype is regulated by sodium/osmolar input.

Combination of gonadal steroid treatment and peripheral nerve grafting results in a peripheral motoneuron-like pattern of beta II-tubulin mRNA expression in axotomized hamster rubrospinal motoneurons.

Rubrospinal motoneurons (RSMN) represent a population of androgen receptor-containing central motoneurons in rodents. In this study, the ability of testosterone propionate (TP), alone or in conjunction with a peripheral nerve graft (PNG), to alter the molecular program of injured RSMN was accomplished using betaII-tubulin cDNA probes and quantitative in situ hybridization (ISH). Initial fluoro-gold labeling experiments following a T1 hemisection established that, as in the rat, the hamster rubrospinal system is essentially crossed and that injured RSMN concentrate in the ventrolateral region of the red nucleus. In the second experimental series, adult gonadectomized male hamsters were subjected to a right T1 hemisection, with half of the operated animals immediately subcutaneously implanted with 1 10 mm TP Silastic capsule and the other half sham implanted. In a third experimental series, animals were subjected to T1 hemisection, followed by transplantation of a predegenerated autologous segment of peripheral nerve. Half of the animals in each group received TP implants at the time of spinal cord injury and PNG. Postoperative times were 2, 7, and 14 days (dpo). Quantitative ISH was performed using a betaII-tubulin-specific (33)P-labeled cDNA probe, emulsion autoradiography, and computerized image analysis for grain counting. Injury alone resulted in a short-lived increase in betaII-tubulin mRNA expression in the RSMN at 2 dpo, with a significant decline to well below control values at 7 and 14 dpo. TP treatment or PNG alone attenuated, but did not prevent, the down-regulation of betaII-tubulin mRNA. In contrast, the combination of TP with a PNG sustained the injury-induced increase in betaII-tubulin mRNA levels throughout the postoperative period of 2, 7, and 14 dpo. The synergistic effects of the two treatment strategies confirm the importance of targeting multiple aspects of the injury response for therapeutic intervention.

Glutamate transporter expression in astrocytes of the rat dentate gyrus following lesion of the entorhinal cortex.

The glutamate transporters GLT-1 and GLAST localized in astrocytes are essential in limiting transmitter signalling and restricting harmful receptor overstimulation. To show changes in the expression of both transporters following lesion of the entorhinal cortex (and degeneration of the glutamatergic tractus perforans), quantitative microscopic in situ hybridization (ISH) using alkaline-phosphatase-labelled oligonucleotide probes was applied to the outer molecular layer of the hippocampal dentate gyrus of rats (termination field of the tractus perforans). Four groups of rats were studied: sham-operated controls, and animals 3, 14 and 60 days following unilateral electrolytic lesion of the entorhinal cortex. The postlesional shrinkage of the terminal field of the perforant path, ipsilateral to the lesion side, was determined and considered in the evaluation of quantitative ISH data. Statistical analysis revealed that ipsilateral to the lesion side there was a significant decrease of the GLT-1 mRNA at every postlesional time-point and of the GLAST mRNA at 14 and 60 days postlesion. The maximal decrease was approximately 45% for GLT-1 and approximately 35% for GLAST. In the terminal field of the perforant path contralateral to the lesion side, no significant changes of ISH labelling were measured. The results were complemented by immunocytochemical data achieved using antibodies against synthetic GLT-1 and GLAST peptides. In accordance with ISH results, there was an obvious decrease of GLT-1 and GLAST immunostaining in the terminal field of the perforant path ipsilateral to the lesion side. From these data we conclude that, following a lesioning of the entorhinal cortex, the loss of glutamatergic synapses in the terminal field of the perforant path resulted in a strong downregulation of glutamate transporters in astrocytes. The decrease of synaptically released glutamate or of other neuronal factors could be involved in this downregulation.

Expression of Type 2 Iodothyronine Deiodinase in Hypothyroid Rat Brain Indicates an Important Role of Thyroid Hormone in the Development of Specific Primary Sensory Systems

Thyroid hormone is an important epigenetic factor in brain development, acting by modulating rates of gene expression. The active form of thyroid hormone, 3,5,3'-triiodothyronine (T3) is produced in part by the thyroid gland but also after 5'-deiodination of thyroxine (T4) in target tissues. In brain, approximately 80% of T3 is formed locally from T4 through the activity of the 5'-deiodinase type 2 (D2), an enzyme that is expressed mostly by glial cells, tanycytes in the third ventricle, and astrocytes throughout the brain. D2 activity is an important point of control of thyroid hormone action because it increases in situations of low T4, thus preserving brain T3 concentrations. In this work, we have studied the expression of D2 by quantitative in situ hybridization in hypothyroid animals during postnatal development. Our hypothesis was that those regions that are most dependent on thyroid hormone should present selective increases of D2 as a protection against hypothyroidism. D2 mRNA concentration was increased severalfold over normal levels in relay nuclei and cortical targets of the primary somatosensory and auditory pathways. The results suggest that these pathways are specifically protected against thyroid failure and that T3 has a role in the development of these structures. At the cellular level, expression was observed mainly in glial cells, although some interneurons of the cerebral cortex were also labeled. Therefore, the T3 target cells, mostly neurons, are dependent on local astrocytes for T3 supply.

Expression of MCH and POMC Genes in Rainbow Trout (Oncorhynchus mykiss) during Ontogeny and in Response to Early Physiological Challenges

The expression of the neuropeptide melanin-concentrating hormone (MCH) in two groups of hypothalamic neurones (NLT- and LVR-MCH neurones), and POMC in the pituitary corticotropes and melanotropes, has been examined in rainbow trout larvae using immunocytochemistry and quantitativein situhybridization. The aim was to establish at what stage in ontogeny these cells first respond to two physiological challenges—background color and stress. Trout reared in black or white trays showed adaptive skin pigmentary changes at 10 days posthatching, when fish in a pale environment abruptly exhibited melanin aggregation from a prior dispersed state, although the pigment cells were already competent to respond to adrenalin and MCHin vitroat 3 days. Immunoreactive MCH was detectable in the neurohypophysis at hatching and MCH mRNA in the NLT-MCH neurones (which project to the pituitary) was enhanced at 7 days in the white-reared trout. Immunostainable POMC was also present in the pars intermedia at hatching but their POMC mRNA was unaffected by tank color until 28 days, when it was enhanced in the black-reared trout. It is suggested that early pigment concentration depends on neural signals from the sympathetic nervous system in conjunction with MCH from the NLT rather than on a reduction in αMSH secretion from the pars intermedia. MCH mRNA in the LVR-MCH neurones was increased on a pale environment only 28 days after hatching, suggesting that these cells play little role in the early adaptive pigment response. Previous studies on the ontogeny of cortisol secretion indicate the hypothalamopituitary–interrenal axis can respond to stress by about 14 days. However, the pituitary ACTH cells showed no stress-induced changes in POMC mRNA until 28 days. ACTH release may therefore be dissociated from POMC transcription in the early stages of development. The LVR- and NLT-MCH neurones were both stimulated by stress, LVR-MCH mRNA responding by 14 days and NLT-MCH mRNA by 21 days. Melanotrope POMC mRNA was reduced by stress but the physiological significance of this is not known.

Estrogen Regulates Neurofilament Gene Expression in Adult Female Rat Dorsal Root Ganglion Neurons

Recently, adult female dorsal root ganglion (DRG) neurons were shown to express the estrogen receptor gene and to bind estrogen. This gene expression and binding is hormone dependent. Moreover, in a subpopulation of DRG neurons, the estrogen receptor is colocalized with high-affinity (trkA) and low-affinity (p75NGFR) receptors for nerve growth factor (NGF). In this NGF-responsive subpopulation of DRG neurons, estrogen regulates expression of the NGF receptor genes and may increase the sensitivity of these cells to the neurotrophin. The present study tested the hypothesis that neurofilament gene expression, which is regulated by NGF in these cells, is dependent on hormone status. In this study, ovariectomized (OVX) rats received either long-term physiological estrogen (conjugated estrogens; Premarin, Wyeth-Ayerst) replacement (low dose) or 10 times physiological levels (high dose). Quantitativein situhybridization with an RNA probe for the 68-kDa neurofilament mRNA revealed a significant dose-dependent effect of Premarin on DRG neurofilament gene expression. In OVX animals receiving low-dose Premarin replacement therapy the mean steady-state 68-kDa mRNA level was as high as 4 times that of untreated OVX rats. High-dose therapy increased the mean 68-kDa neurofilament steady-state mRNA level to as much as six-fold that observed in untreated OVX animals. The estrogen-dependent upregulation of neurofilament gene expression appeared to occur in all DRG neurons, rather than in a subpopulation of those cells. These data suggest an important role for estrogen in the maintenance and function of primary sensory neurons. Whether estrogen directly regulates neurofilament gene expression or indirectly regulates it by increasing DRG neuronal sensitivity to neurotrophins or other growth factors remains to be determined.

Aldose Reductase and IGF-I Gene Expression in Aortic and Arteriolar Smooth Muscle during Hypo- and Hyperinsulinemic Diabetes

Two genes whose expression is likely to be altered during diabetes mellitus are aldose reductase (AD) and insulin-like growth factor-I (IGF-I). We proposed that gene expression of AD is increased in vascular smooth muscle during diabetes mellitus due to hyperglycemia, while IGF-I expression is decreased in insulin-deficient diabetes and elevated in insulin-resistant diabetes. The mRNA for both was measured in the renal glomerulus, in the vascular smooth muscle of large arterioles from the brain, kidney, and small intestine, and in the aorta of hypoinsulinemic streptozotocin (STZ)-treated rats and hyperinsulinemic Zucker diabetic fatty (ZDF) rats. Quantitativein situhybridization was used to determine variations in expression. Expression of the AD gene was unchanged in STZ and ZDF rats, except for a decrease of about 50% in glomeruli and renal smooth muscle of STZ diabetic rats. Expression of IGF-I generally decreased in vascular smooth muscle of insulin-depleted STZ diabetic rats, but was normal in hyperinsulinemic ZDF rats. The data indicate that decreased expression of the AD gene is a specific problem in renal vascular smooth muscle and glomeruli in the insulin-depleted STZ model of diabetes. The expression of the IGF-I gene in vascular muscle was decreased in hypoinsulinemic diabetic animals, but did not increase in hyperinsulinemic diabetic rats.

Regulation of α7Nicotinic Acetylcholine Receptors in the Developing Rat Somatosensory Cortex by Thalamocortical Afferents

Distributions of alpha7 nicotinic acetylcholine receptor (nAChR) mRNA and [125]alpha-bungarotoxin (alpha-BTX) binding sites in the developing rat somatosensory cortex were characterized in relation to acetylcholinesterase (AChE) histochemical staining of thalamocortical terminals to investigate the role of this receptor in cortical development. Using quantitative in situ hybridization and receptor autoradiography, elevated levels of mRNA and binding-site expression were first detected at post-natal day 1 (P1) in deep and superficial layers, just beneath the AChE-stained thalamocortical terminals. Onset of expression occurred approximately 1 d after ingrowth of AChE-stained thalamocortical afferents. By P5, mRNA and binding-site expression exhibited a disjunctive, barrel-like pattern in layer IV and, more clearly, in layer VI. The mRNA and binding-site expressions peaked at approximately 1 week postnatal and then declined to adult levels. Unilateral electrolytic or cytochemical lesions placed in the thalamic ventrobasal complex at P0 (just as thalamocortical afferents are innervating the cortex) and at P6 (when the somatotopic map is well established) resulted in a marked reduction of alpha7 nAChR mRNA and [125]alpha-BTX binding-site levels in layers IV and VI, indicating their regulation by thalamocortical afferents. With P6 lesions, this reduction was observed as early as 6 hr postlesion. These results suggest that alpha7 nAChRs are localized primarily on cortical cells in rat somatosensory cortex and provide further evidence for thalamocortical influence on cortical ontogeny. These data also suggest a role for cholinergic systems during a critical period of cortical synaptogenesis.

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of fluorescence in situ hybridization signals by image cytometry.

In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.