For Fluorescence In Situ Hybridization Research Articles

A Hybrid Approach to the 3D High Precision Position Measurement and Particle Tracking in Human Cell Nuclei

In this report, we present a detection and tracking system for fluorescent particles inside a cell nucleus. We present the application of this system to a real biological problem where the same human cell nucleus is scanned in three different states: living, fixed and after a treatment used for fluorescence in situ hybridization (FISH). The change in distances between internal sub-chromosomal structures was analyzed.

Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries.

A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated T(d) values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries.

Microbial community changes during organic solid waste treatment analyzed by double gradient-denaturing gradient gel electrophoresis and fluorescence in situ hybridization.

The bacterial community present during semicontinuous treatment of organic solid waste under alkaline and high-temperature conditions was studied. PCR-amplified 16S rDNA fragments were analyzed by double gradient-denaturing gradient gel electrophoresis (DGGE). The band pattern was stable during the steady state of the treatment phase, and the major bands resulting from individual treatments had the same DNA sequence with good reproducibility. No sequence in the DNA database of isolated bacteria showed close similarity to this sequence, the closest relative being Bacillus licheniformis with less than 97% similarity. The conditions for fluorescence in situ hybridization (FISH) were determined without the need to obtain extracts of the bacterial cells. An oligonucleotide probe was designed to detect the microorganisms found in the DGGE analysis. FISH analysis showed that the bacterium corresponding to the major bands accounted for 30% of the total eubacterial cell count at the steady state. These results indicate that this bacterium is a key microorganism in the biodegradation process.

A molecular cytogenetic map of sorghum chromosome 1. Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes.

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.

Morphometric and cytogenetic characteristics of testicular germ cells and Sertoli cell secretory function in men with non-mosaic Klinefelter's syndrome.

Klinefelter's syndrome is the most frequent chromosomal abnormality in infertile men. In this study, the chromosomes of round spermatids and spermatogonia/primary spermatocytes from men with non-mosaic Klinefelter's syndrome were examined, together with the Sertoli cell secretory function and sperm morphometry. Twenty-four men with non-mosaic Klinefelter's syndrome and nine men with obstructive azoospermia underwent therapeutic testicular biopsy. When spermatozoa in the final filtrate were present, they were processed for sperm morphometry or ICSI. Sperm morphometry was evaluated by the maximal length and width of the sperm head, the length of the midpiece and the ratio of the acrosomal region to the total surface area of the head. When round spermatids were present, they were processed for fluorescent in-situ hybridization (FISH). FISH was also applied to fragments of seminiferous tubules. Sertoli cell secretory function was measured by the amount of androgen binding protein (ABP) secreted in vitro. More than 93% of the evaluated round spermatids were normal. The proportions of 24,XY and of 24,XX round spermatids to the total number were significantly larger in men with Klinefelter's syndrome than in obstructed azoospermic men. Men with Klinefelter's syndrome who had spermatozoa in their testicular tissue (n = 12) were positive for both 46,XY and 47,XXY spermatogonia in their seminiferous tubules. In contrast, men with Klinefelter's syndrome without spermatozoa in their testicular tissue (n = 12) were positive for 47,XXY spermatogonia but negative for 46,XY spermatogonia in their seminiferous tubules. ABP profiles were significantly smaller in men with Klinefelter's syndrome who were negative for spermatozoa compared with men who were positive. Four pregnancies were achieved and five healthy babies were born. This study suggests that few 46,XXY spermatogonia undergo meiosis with an XX pairing and a Y univalent type of pairing. Hyperhaploid round spermatids (24,XY and 24,XX) may be produced by meiosis of 47,XXY spermatogonia. Men with Klinefelter's syndrome who are negative for testicular spermatozoa have a greater degree of Sertoli cell secretory dysfunction compared with men with Klinefelter's syndrome who are positive for spermatozoa. There are several defects in sperm morphometry with functional significance in men with Klinefelter's syndrome.

The Microbial Community Composition of a Nitrifying-Denitrifying Activated Sludge from an Industrial Sewage Treatment Plant Analyzed by the Full-Cycle rRNA Approach

The composition of the microbial community present in the nitrifying-denitrifying activated sludge of an industrial wastewater treatment plant connected to a rendering facility was investigated by the full-cycle rRNA approach. After DNA extraction using three different methods, 94 almost full-length 16S rRNA gene clones were retrieved and analyzed phylogenetically. 59% of the clones were affiliated with the Proteobacteria and clustered with the beta- (29 clones), alpha- (24), and delta-class (2 clones), respectively. 15 clones grouped within the green nonsulfur (GNS) bacteria and 11 clones belonged to the Planctomycetes. The Verrucomicrobia, Acidobacteria, Nitrospira, Bacteroidetes, Firmicutes and Actinobacteria were each represented by one to five clones. Interestingly, the highest 'species richness' [measured as number of operational taxonomic units (OTUs)] was found within the alpha-class of Proteobacteria, followed by the Planctomycetes, the beta-class of Proteobacteria, and the GNS-bacteria. The microbial community composition of the activated sludge was determined quantitatively by using 36 group-, subgroup-, and OTU-specific rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH), confocal laser scanning microscopy and digital image analysis. 89% of all bacteria detectable by FISH with a bacterial probe set could be assigned to specific divisions. Consistent with the 16S rRNA gene library data, members of the beta-class of Proteobacteria dominated the microbial community and represented almost half of the biovolume of all bacteria detectable by FISH. Within the beta-class, 98% of the cells could be identified by the application of genus- or OTU-specific probes demonstrating a high in situ abundance of bacteria related to Zoogloea and Azoarcus sensu lato. Taken together, this study provides the first encompassing, high-resolution insight into the in situ composition of the microbial community present in a full-scale, industrial wastewater treatment plant.

Routine acid decalcification of bone marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer.

Production of paraffin-section material from tissue samples that contain bone requires decalcification. Techniques such as acidic decalcification or EDTA chelation are suitable methods. Acid decalcification is generally quicker than EDTA chelation but studies have suggested that it may result in hydrolysis of DNA. Here we show that limited acid decalcification (less than 24 hr) in 5% formic acid can preserve DNA sufficient for fluorescent in situ hybridization (FISH) or comparative genomic hybridization (CGH) and that prolonged 10% formic acid decalcification results in failure of FISH and only limited retrieval of DNA for CGH studies.

Improvement of Ciliate Identification and Quantification: a New Protocol for Fluorescence In Situ Hybridization (FISH) in Combination with Silver Stain Techniques

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.

ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III (May 2001).

The ESHRE PGD Consortium was formed in 1997 to undertake a long-term study of the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD). Here, the third report of the ESHRE PGD Consortium is presented, collating data received from 25 centres on referrals, cycles, pregnancies and babies born after PGD. The second report, published in December 2000, reported on 886 referrals, a total of 1318 started cycles (of which 465 for aneuploidy screening, 386 for fluorescence in-situ hybridization (FISH) and 385 for PCR going beyond oocyte retrieval), 163 pregnancies and 162 children born. This year, 675 referrals from 12 centres were added giving a total of 1561 referrals, 370 regular PGD cycles, 334 PGD-aneuploidy screening (PGD-AS) cycles and 78 cycles for social sexing from 24 centres and 215 pregnancies and 117 babies from 12 centres. Because more in-depth information was asked for the cycles, this year's data will be shown separately as well as cumulatively. One striking feature of this year's data collection is the appearance of the first data for gender screening on preimplantation embryos for social reasons. The ethical concerns regarding social sexing will be discussed, as well as the forthcoming changes in timing of data collection. When the data collection was discussed at the last meeting of the members of the ESHRE PGD Consortium in Lausanne, Switzerland (June, 2001), it appeared that the current system of data collection, although yielding results very quickly, showed fundamental flaws. The ESHRE PGD Consortium Steering Committee intends to remedy to these problems, on the one hand by introducing a new type and timing of data collection, and on the other hand by re-analysing and correcting the data which have already been sent in during the past 4 years.

Conservation of avian Z chromosomes as revealed by comparative mapping of the Z-linked aldolase B gene

A chicken Z-linked BAC probe containing the aldolase B gene was used for fluorescence in-situ hybridization (FISH) mapping in four different avian species. The biotinylated BAC clone showed distinct unique hybridization sites on the structurally different Z chromosomes. This result, together with previous data, lends credence to the notion that, despite undergoing structural rearrangements, the gene content of the avian Z chromosome remained conserved during evolution. Our study also demonstrates the feasibility of using large genomic clones for comparative mapping of Z-linked genes in birds.

Microwave decondensation and codenaturation: a new methodology to maximize FISH data from donors with very low concentrations of sperm

To increase analyzable sperm numbers when dealing with semen samples from donors with extremely low quantities of sperm, a microwave decondensation/codenaturation technique for fluorescence in situ hybridization (FISH) was developed and results were compared to those obtained from conventional FISH techniques. There was no significant difference between the frequencies of disomy and diploidy obtained using the two techniques.

Conserved synteny and gene order difference between human chromosome 12 and pig chromosome 5

A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs). These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2). Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison. Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1. These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases. In addition, the human YACs were FISH mapped on pig chromosomes. This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel. While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig. In addition, it was also possible to determine the position of the synteny breakpoint.

Delineation of a 150-kb breakpoint cluster in benign thyroid tumors with 19q13.4 aberrations

Structural rearrangements involving the long arm of chromosome 19 characterize a cytogenetic subgroup of benign thyroid tumors and constitute one of the most frequent specific chromosome abnormalities in epithelial tumors. Recently, we have been able to narrow down the breakpoint region affected in two cell lines to a region covered by a single PAC clone. Close to that region a candidate gene has been identified which we tentatively referred to as RITA (Rearranged In Thyroid Adenomas) now named ZNF331 according to HUGO nomenclature. However, the results had been obtained on two cell lines only making it necessary to extend the studies to a larger number of tumors including primary material. Herein, we have used four further primary tumors showing translocations involving 19q13 for fluorescence in situ hybridization (FISH) mapping studies using a variety of molecular probes from a 470-kbp cosmid/BAC contig. Ten new STSs were characterized and physically mapped within an EcoRI restriction map. The results enabled us to define an approximately 150-kbp breakpoint cluster region of the 19q13 aberrations in benign thyroid tumors flanked by two newly established STS markers.

Chromosomal location and evolution of a satellite DNA family in seven sturgeon species.

The Hind III satellite DNA family, isolated from the Acipenser naccarii genome, was used as a probe for fluorescent in-situ hybridization (FISH) on the karyotype of seven sturgeon species, six belonging to the genus Acipenser and one to Huso. All species except one (A. sturio) exhibit from 8 to 80 chromosome hybridization signals, mainly localized at the pericentromeric regions. Eight chromosomes with weak hybridization signals are present in H. huso and A. ruthenus, which are characterized by a karyotype with about 120 chromosomes. The species with 240-260 chromosomes, A. transmontanus, A. naccarii, A. gueldenstaedtii, and A. baerii, show from 50 to 80 signals, prevalently localized around centromeres. Moreover, A. transmontanus and A. gueldenstaedtii show from 4 to 8 chromosomes with a double signal. The phylogenetic and evolutionary relationships among sturgeon species are discussed on the basis of number and morphology of signal-bearing chromosomes and on the localization of signals.

Major histocompatibility complex and immunoglobulin loci visualized by in situ hybridization on Xenopus chromosomes

A technique for fluorescent in situ hybridization (FISH) on chromosomes of the amphibian Xenopus laevis is described. Positive results were obtained with cDNA probes of about 1 kb when at least three adjacent copies of the gene are present. The immunoglobulin heavy chain locus is in the centre of the long arm of chromosome 1. Previously, family studies showed that bona fide MHC class Ib genes segregated independently. Now we show that MHC class II α and β genes and class Ib genes are on the same acrocentric chromosome, with MHC in the middle of the long arm, the class Ib complex (XNC) at the tip or the same arm. Each locus or complex is found on only one pair of chromosomes confirming the diploidization of these genes in the pseudotetraploid X. laevis.

Risks of in-vitro production of cattle and swine embryos: aberrations in chromosome numbers, ribosomal RNA gene activation and perinatal physiology.

In cattle, in-vitro production (IVP) of embryos has become a standardized technique; however, increased frequencies of calving problems and larger calves have been reported. In swine, IVP has resulted in only a limited number of piglets. In this paper we present information on cattle and swine embryos produced in vitro by oocyte maturation, fertilization and further embryo culture to the blastocyst stage in vitro. Control in-vivo developed embryos were collected after superovulation. The cattle embryos were processed for fluorescence in-situ hybridization (FISH) with two chromosome-specific probes to detect numerical chromosome aberrations. The swine embryos were processed for transmission electron microscopy and immunocytochemistry with an antibody against RNA polymerase I [essential for ribosomal RNA (rRNA) gene transcription] in order to highlight the post-fertilization development of the nucleolus as a marker for rRNA gene activation. The FISH analyses of the cattle embryos revealed that 72% of IVP blastocysts were mixoploid, i.e. contained both diploid and polyploid cells, versus 25% in vivo. Chromosome abnormalities were observed from the 2-cell stage onwards. The immunocytochemical analyses of the swine embryos revealed that during in-vivo development, RNA polymerase I became localized to multiple foci in the developing nucleoli late during the 4-cell stage. This focal localization of RNA polymerase I was not observed in IVP embryos. In conclusion, IVP embryos may display aberrations in chromosome numbers and rRNA gene activation. The significance of these deviations for fetal and perinatal viability, however, remains unknown. The survival of most calves derived from IVP indicates that a considerable number of these embryos are able to compensate for the adverse effects of the in-vitro procedures.

Effects of Hormones on Spermatogenesis in Men with Histological Diagnosis of Spermatogenic Arrest at the Primary Spermatocyte (PS) Stage or Sertoli Cell-Only Syndrome (SCOS)

Objectives: Men with absence of spermatozoa in the therapeutic testicular biopsy (TTB) material represent the most difficult cases of non-obstructive azoospermia (NOA). Even when alive round spermatids (RSs) are present in the TTB samples of the latter men, the outcome of ooplasmic injections of RSs rarely alleviates the couple’s infertility. We evaluated the effects of a combined administration of Profasi (Serono Co.) and Gonal-F (Serono Co.) on testicular spermatogenic and steroidogenic function. Design: Diagnostic testicular biopsy (DTB; hematoxylin-eosin stain) outcome and TTB (mincing testicular tissue and processing it for assisted reproduction trial) outcome were compared before and after hormonal treatment in three groups of NOA-men. Materials and Methods: All men had peripheral serum LH and FSH values less than twice the upper normal level of each respective hormone. TTB was performed at least four months after the performance of DTB. At the time of DTB, intratesticular testosterone (ITT) was measured, whereas, at the time of TTB, fractions of dispersed cells were processed for fluorescent in situ hybridization (FISH) techniques or observation via transmission electron microscope (TEM). Gonal-F (50 IU three times a week) and Profasi (2,500 IU twice a week) were administered for 12–24 months in a) 21 men whose DTB showed arrest at the PS stage and the TTB revealed absence of RSs and spermatozoa (group A), b) 7 men whose DTB demonstrated arrest at the PS stage and the TTB was positive for RSs but negative for spermatoa/elongating/elongated spermatids (group B), and c) 5 men whose DTB showed SCOS and the TTB demonstrated few PSs but not RSs (group C). New DTB, TTB, FISH, and TEM techniques were performed at the end of the treatment. Results: At the end of the homonal treatement, DTB images showed the same level of spermatogenic arrest as prior to treatment in all men of groups A, B, and C. Peripheral serum testosterone increased in all the participants. Observations of TTB samples post-treatment demonstrated few RSs in 8 men of group A. One of the latter 8 men showed spermatozoa, as well. No RSs were observed in TTB-samples of group C post-treatment. TEM showed elongating or elongated spermatids having tail length 0.004–0.047 mm in the TTB materials of 5 men of group B post-treatment. However, testicular spermatozoa were found in only two of the latter men. When the groups A and B were analyzed together it was found that the 13 men who responded to the treatment (development of RSs in the second TTB for group A; spermatid elongation in the second TTB for group B) had (prior to the hormonal treatment) significantly lower (P<0.05; Wilcoxon’s test) ITT profiles than the 15 remaining men who did not respond to the treatment. There was a large variation in ITT profiles prior to the treatment in groups A and B (taken together the groups A and B: 76–2675 ng/g testis). Four out the five men of group B who responded to the treatment had relatively low ITT profiles (less than 300 ng/g testis) pre-treatment. Conclusions: Hormonal treatment in men with early maturation arrest stimulates spermatogenesis in a clinically significant percentage (46%). Prior to the hormonal administration, the presence of RSs in the TTB of men with histological diagnosis of early maturation arrest or small ITT levels indicates good prognosis for response to the treatment. Hormonal treatment in SCOS-men without foci of RSs/spermatozoa is not recommended. This research was supported by a grant from the JG Fertility Institute.

Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes.

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083(T). The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.

Molecular analysis and development of 16S rRNA oligonucleotide probes to characterize a diclofop-methyl-degrading biofilm consortium.

Genomic DNA from nine individual bacteria, isolated from a diclofop-methyl-degrading biofilm consortium, was extracted for genetic characterization. The degradation of diclofop-methyl produces metabolites that are known intermediates or substrates for bacteria that degrade a variety of chlorinated aromatic compounds. Accordingly, oligonucleotide primers were designed from specific catabolic genes for chlorinated organic degradation pathways, and tested by PCR to determine if these genes are involved in diclofop-methyl degradation. DNA homology between the PCR products and the known catabolic genes investigated by Southern hybridization analysis and by sequencing, suggested that novel catabolic genes are functioning in the isolates. Specific fluorescent oligonucleotides were designed for two of the isolates, following 16S rDNA sequencing and identification of each of the isolates. These probes were successfully used for fluorescent in situ hybridization (FISH) studies of the two isolates in the biofilm consortium.

Quantitative assessment of the alteration of chromatin during the course of FISH procedures

Background: DNA denaturation, required for fluorescent in situ hybridization (FISH) experiments, is likely to induce chromatin alterations. Only few attempts have been made to quantify the extent of these perturbations. We propose a quality-control approach based on image analysis to monitor the effect of a procedure commonly used in FISH experiments. Methods: Using DAPI as a probe, the same nuclei were successively imaged with a CCD camera after fixation, after permeabilization, and after thermal denaturation and hybridization with a centromeric probe. The modifications of the staining pattern were analyzed. Volumes of the FISH signals were measured using confocal imaging. Results: DAPI staining combined with image analysis proved to be a sensitive tool to visualize the effects of different treatments used in FISH experiments. Permeabilization of nuclei after fixation has only limited impact on the chromatin. On the contrary, the denaturation procedure modifies the staining of DNA by DAPI, as well as the underlying chromatin structure as assessed by the increase of FISH signal volume with denaturation time. The protocol that involves a pre-fixation permeabilization step results in a more severe loss of chromatin structure. Conclusions: Our results clearly show that analysis of alterations of DAPI staining patterns is a useful monitoring tool to control and standardize hybridization procedures. Cytometry 36:96‐101, 1999. r 1999 Wiley-Liss, Inc. Key terms: DAPI; chromatin texture; FISH; fluorescence microscopy; image analysis