Histochemical journal 14, 333-344 (1982)
`
`
`
`Spreading and staining of human metaphase
`chromosomes on aminoall<ylsilane—treated glass
`slides
`
`A. C. VAN PROOIJEN-KNEGT, A. K. RAAP, M. ]. M. VAN DER
`BURG, J. VROLIJK and M. VAN DER PLOEG
`
`Department of Histochernistry and Cytochemistry, Sylvius Laboratories, Wassenaarseweg 72, NL-2333 AL
`Leiden, The Netherlands
`
`Received 25 June 1981
`
`
`
`Summary
`
`The properties of aminoall<ylsilane—treated glass slides for the preparation of metaphase spreads
`and their staining quality have been studied and compared with those of slides which had only
`been cleaned in ethanol/ether. The parameters investigated were:
`(1)
`the average area of
`metaphases from cultures of blood from both healthy donors and haematology patients; (2) the
`influence of the positively charged ‘coating’ on the quality of quinacrine- and Giemsa-banding
`patterns; (3) non-specific background staining for these banding methods; (4) the number of
`metaphases as compared to the number of interphase cell nuclei per area of preparation; and (5)
`the Feulgen—staining intensities of chromosomes and chicken erythrocyte nuclei.
`The quality of metaphase preparations and the differential staining of chromosomes is better
`on aminoalkysilane-treated glass slides than that of preparations on routinely cleaned normal
`microscope slides. In the preparations on aminoalkylsilane-treated slides, the distribution of the
`cells over the glass surface is more homogeneous; and no influence could be detected on the‘
`relative frequency of metaphases as compared to the number of non—divided cell nuclei; the
`average area per metaphase is increased by about 10% and consequently the number of
`overlapping chromosomes is decreased.
`'
`Preparations on aminoalkylsilane-treated glass, after Q—, G— and DAPI-banding procedures,
`always showed less binding of the staining compounds to the glass slide (a cleaner background)
`than those on routinely cleaned microscope glass slides. The Feulgen-pararosaniline staining
`intensities of human metaphase chromosomes and chicken erythrocyte nuclei are the same on
`aminoalkylsilane-treated slides and on routinely cleaned glass
`slides. Furthermore,
`the
`reproducibility and constancy of quinacrine banding was improved by development of an
`equilibrium staining method which does not require a washing procedure. The medium,
`containing
`0.002% quinacrine,
`allows optimal
`staining results
`to
`be obtained for
`microphotography purposes within 30 min of staining (for visual inspection at least 90 min is
`required) and is used as the embedding medium.
`,
`A
`In combination with aminoalkylsilane—treated glass slides,
`this procedure leads to a clean
`background and reproducible banding patterns of excellent quality, the results being better and
`more constant than those of methods described before.
`
`0018-2214,/82/020333-12$03.94/O © 1982 Chapman and Hall Ltd.
`Page 1 of 12
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`
`334
`
`Introduction
`
`VAN PROOIJEN-KNEGTETAL.
`
`The purpose of the present study was to improve the quality and the reproducibility of
`banding procedures for human metaphase chromosomes. Special attention was paid to
`two parameters: non-specific background staining in the microscope slides, and the
`heterogeneous staining which readily occurs during the washing procedure after
`quinacrine staining.
`The background staining that occurs when compounds like quinacrine, quinacrine
`mustard, DAPI and Giemsa are applied for the differential staining of metaphase
`chromosome preparations is the result of a strong binding capacity of these dyes to
`glass. This binding could be caused by the electrostatic attraction between the negative
`silicate groups of glass and the positive groups of the basic dye compounds and, based
`on this hypothesis, it was expected that coating the glass surface with positive groups,
`such as amino groups would diminish this type of background staining.
`To overcome the problem of background staining, glass slides were treated with
`3—aminopropyltriethoxysilane which provides the glass surface with alkylamine groups.
`It was found that aminoalkylsilane-treated slides,
`in accordance with expectations,
`showed hardly any background staining after quinacrine or Giemsa incubation. Two
`favourable side effects of
`the aminoalkylsilane treatment were observed: better
`spreading of the metaphase plates and a more homogeneous distribution of the cells
`over the slide.
`
`Heterogeneity in quinacrine staining results readily arises during the differentiation
`procedure necessary in quinacrine staining methods published so far. Rinsing is critical,
`and has to be done briefly in order to prevent the removal of all quinacrine, and this may
`lead, when not performed carefully, to preparations in which parts are still overstained
`while other parts have already lost too much of the dye to show a clear banding pattern.
`To solve the problem of heterogeneous staining intensities among the metaphases, a
`quinacrine staining procedure was developed in which the concentration of the dye
`compound is so low that it is not necessary to wash the excess of quinacrine away after
`staining. Due to the lower dye concentration in the staining medium, longer staining
`times are necessary, but equilibrium~stained preparations embedded in the staining
`solution clearly show a more constant high quality banding pattern all over the
`
`preparation.
`
`Materials and methods
`
`AMINOALKYLSILANE-TREATED GLASS SLIDES
`
`(1971), microscope glass slides were
`Following the procedure described by Robinson at all.
`cleaned by incubation overnight
`in a 10% solution of Extran MAO1 (alkalisch, E. Merck,
`Darmstadt, Germany) in distilled water, rinsing with hot (600 C) tap water and with distilled
`water and drying at 80° C. These slides were then incubated for 16 h in a 2% solution of
`3-aminopropyltriethoxysilane (Aldrich Europe, Beerse, Belgium) in dry acetone. Thereafter, the
`slides were rinsed in acetone and two changes of distilled Water and stored in 0.02% NaN3 in
`
`Page 2 of 12
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`
`

`
`Metaphase chromosomes on aminoalkylsilane-treated slides
`
`335
`
`distilled water. Immediately before use, the slides were rinsed in distilled water and air dried.
`Slides could be stored for up to six weeks without losing their favourable properties such as the
`reduced binding of basic dye compounds. Results obtained with slides which had been stored dry
`at room temperature were not significantly different when these slides were used within two
`weeks after preparation.
`
`CHROMOSOME PREPARATIONS
`
`Metaphase preparations on microscope slides were prepared from human total blood cultures as
`described in detail by Bosman et al . (1975). By this method chromosome preparations on glass are
`obtained which show a relatively low protein background and a good rnetaphase spreading. After
`the hypotonic treatment and centrifugation, the resulting cell pellet is resuspended in the tube
`with the last drops of supernatant. About 5 ml of freshly prepared methanol/acetic acid fixative is
`then gently mixed with the cell suspension by slowly aspirating the contents of the tube into a
`Pasteur pipette already filled with the fixative. The erythrocyte remnants then dissolve and
`repeated replacement of the fixative removes non cellular~bound proteins. The blood samples
`were obtained from healthy volunteers as well as from haematology patients.
`
`STAINING PROCEDURES
`
`Quinacrine staining was performed in two ways: (1) Slides were incubated for 5 min in Mcllvaine
`buffer, pH 4.1, and stained for different periods varying from 10 min to 4 h in an excess of 0. 002%
`quinacrine (G. T. Gurr—Searle, High Wycombe, Bucks, U.K.) dissolved in the buffer. These
`preparations were mounted in the staining medium itself, and sealed with Fluoromount (Gurr).
`(2) After pre—incubation in Mcllvaine buffer, pH 4.1, for 5 min and staining of the preparations in
`a 0.1% quinacrine solution in the buffer during 5 min, the preparations were washed with two
`short buffer rinses and mounted in the Mcllvaine buffer.
`
`DAPI staining
`Chromosome preparations were stained in a 0.3 pg/ml solution of DAPI (Serva, Heidelberg,
`Germany) in Mcllvaine buffer, pH 7.0, for 20 min. Slides were washed with distilled water and
`Mcllvaine buffer, pH 5.5, for 10 s and embedded in the same buffer.
`
`Giemsa banding
`Metaphase preparations were stained following the method described by Sumner et al. (1971)
`with minor modifications. Slides were incubated for 1 h at 65° C in 2xSSC (0.3 M NaCl, 0.03 M
`trisodium citrate, pH 7.0) after which they were rinsed in running demineralized water. Then the
`slides were stained for 15 min in freshly made Giemsa medium [1 part of Giemsa solution (C-.urr’s
`Giemsa R66) and 19 parts of Gurr buffer pH 6.8 (prepared with buffer tablets) filtered through
`prefolded filter paper], washed in running demineralized water, and air dried.
`
`Feulgerz staining
`After fixation in a freshly prepared mixture of methanol, formaldehyde solution 35% (w/V) and
`glacial acetic acid (85 2 10 : 5, by volume) for 1 h at room temperature, chromosome preparations
`were stained with the pararosaniline(SO2) reagent described by Duijndam & Van Duijn (1973).
`The Schiff reagent was prepared according to Graumann (1952-1953) from pararosaniline
`(—’Acridinfrei’, Chroma, Stuttgart, Germany). The stained chromosome preparations were passed
`through an ethanol—xylene dehydration series and were mounted in a mixture of Fluoromount
`and Cargille oil as described by Van der Ploeg et al. (1977).
`
`Page 3 of 12
`Page 3 of 12
`
`

`
`336
`
`VAN PROOl]EN—KNEGTETAL.
`
`FLUORESCENCE MICROSCOPY, MICROPHOTOGRAPHY AND SCANNING
`CYTOPHOTOMETRY
`
`Chromosome preparations were examined with a DIALUX 20 microscope (Leitz GmbH, Wetzlar,
`Germany) provided with epi—illumination and a mercury arc (HBO 200 W, Osram GmbH, Berlin,
`Germany). Visualization of quinacrine fluorescence emission was obtained with a combination of
`a Calflex heat absorption filter (Balzers AG, Lichtenstein) and a band interference filter AL 436
`(Leitz) in the excitation light path; a dichroic mirror (AH 455); and a barrier filter KP 490 (Leitz).
`DAPI fluorescence emission was observed with the same microscopic set-up, using a UG 1
`(Schott & Gen., Mainz, Germany) and a BG 38 (4 mm, Leitz) combination in the excitation beam;
`a dichroic mirror (AH 400, Leitz) and an LP 460 (Schott) as a barrier filter.
`A Neofluar 100 X/1.30 objective (Carl Zeiss, Oberkochen, Germany) was used both for visual
`inspection and for fluorescence photography on 35 mm Kodak High Contrast Copy Film
`(Eastman Kodak Comp., Rochester, New York, U.S.A.)* after pre-exposure of the film emulsion
`as described by Van der Ploeg et al. (1976). The actual exposure time for chromosomes stained
`with quinacrine was 10 s. Photography of DAPI fluorescence emission took 5 s.
`Absorption photography of the Feulgen- or Giemsa-stained preparations was performed as
`described previously (Van der Ploeg at al., 1974a, b) with an AL 559 interference filter (Schott)
`inserted in the illumination beam. Agfa—Ortho 25 professional (Agfa—Gevaert, Antwerp, Belgium)
`was used as photographic emulsion. The exposure times were chosen so that the optical densities
`of the negatives were located on the straight part of the Hurter and Driffield curve of the film
`emulsion.
`
`For the scanning procedure, the negatives were embedded in immersion oil between glass
`slides and sealed with Fluoromount. Interactive scanning of individual No. 2 chromosome images
`was performed using the HIDACSYS-PROFILSCAN or -ARRAYSCAN program (Bosman et al ., 1977; Van
`der Ploeg et al., 1977). The stage-scanner was a CYTOSCAN SMP (Carl Zeiss) interfaced to a
`PDP 11/O4 computer (Digital Equipment, Maynard, Massachusetts, U.S.A.). Sample and line
`separation were 40 pm in the micrographs (which equals a spatial resolution of 0.125 /rm at the
`original specimen plane); light transmittance values were quantized in 512 linear intensity levels,
`which were then converted into absorbance values.
`
`Scanning absorbance measurements of Feulgen—stained chicken erythrocyte nuclei and
`metaphases were performed directly in the preparations with the SMP stage scanner at 560 nm,
`using the FASTSCAN program. Sample and line separation were 0.25 or 0.50 gm; further details
`were as specified for the photographic negative scanning.
`Determination of
`the quality of metaphase spreading was performed directly in the
`Giemsa-stained chromosome preparations with a TAS television scanner (Leitz). For this
`purpose, a computer program was used that differentiated between metaphases, artefacts and
`nuclei, and determined the area of each recognized metaphase in pixels together with the number
`of metaphases and nuclei detected in a scanned area. Determination of the area of a metaphase is
`performed by dilating (Serra, 1974) the areas of its individual chromosomes until finally one
`object is obtained (Fig. 1a), which then is eroded as many times as originally the chromosomes
`had been expanded (Fig. 1b). In each preparation three areas of 10 X 10 microscopic fields (each
`0.105 mmz) were scanned. The areas were taken centrally, 1 cm apart along the long axis of the
`glass slide.
`
`*Because the High Contrast Copy Film is no longer available, we switched recently to Kodak Technical Fan
`Film (ESTAR—AI-I Base) SO-115 with D 19 as the developer. The sensitivity of this film emulsion is higher (the
`actual exposure time for both types of quinacrine-stained preparations is 2 s, for DAPI fluorescence 0.5 s), and
`the grain size is sufficiently fine.
`
`Page 4 of 12
`Page 4 of 12
`
`

`
`Metaphase chromosomes on aminoall<ylsilane—treated slides
`
`337
`
`Fig. 1. The determination of the area of a metaphase. (A) Schematic representation of the image
`resulting after dilation of the thresholded individual chromosomes of a metaphase until one object
`is obtained. (B) The area of the metaphase, which results after erosion of image (A) as many times
`as the individual chromosomes had been dilated.
`
`Results
`
`The quality of quimzcrine staining results
`Visual evaluation was performed ‘blind’ by five cytogeneticists independently on the
`preparations themselves, and also on photographic prints of metaphases which had
`been spread on:
`I
`
`(A) aminoalkylsilane-treated glass slides or;
`(B) microscope glass slides cleaned with ethanol,/ether, after staining with quinacrine,
`using either (I) the ‘routine’ method (0.1%, 5 min, washing), or (II) the equilibrium
`method (0.002%, 2 h).
`
`The results led to the following conclusions:
`
`(1) preparations on 3-aminopropyltriethoxysilane-treated glass always showed less
`background (glass) staining than those on alcohol/ether-“cleaned slides.
`
`Page 5 of 12
`Page 5 of 12
`
`

`
`Page 6 of 12
`
`

`
`Metaphase chromosomes on aminoall<ylsilane—treated slides
`
`339
`
`[
`..»_',J
`I
`“.3 ml.
`.-.
`la-\
`. 2. — I
`"ma
`
`'
`
`3'--.;.-..'
`
`-' 3
`
`
`
`_0
`‘n
`—:..
`
`iii‘
`
`Fig.3. Giemsa-stained metaphase preparations on aminoalkylsilane-treated glass (a) and on
`alcohol/ether-cleaned slides
`The decreased background staining in the first
`type of
`preparation can already be observed macroscopically, especially in the ground end of the slide.
`
`(2) the results after routine staining sometimes showed Variations in quality as expected
`because of the washing procedure. The overall quality of the banding pattern(s) of
`the equilibrium—stained preparations was more uniform and always better than that
`in the routinely stained preparations.
`(3) the distribution of cells and metaphases over the slides is more homogeneous than
`in glass slides cleaned with ethanol/ether.
`(4) the chromosomes of metaphase spreads on aminoalkylsilane-treated glass slides
`seemed to be spread over larger areas than those on ethanol/ether-cleaned glass
`(Fig. 2).
`
`Giemsa—banded metaphases from both types of preparations were similarly evaluated
`visually.
`In all aminoalkylsilane-treated glass preparations,
`the overall background
`staining intensity is lower, as can already be observed macroscopically (Fig. 3). The
`quality of the banding patterns was good in both the routinely cleaned and the
`amin0alkylsilane—treated glass preparations, and no significant difference could be
`detected.
`
`The results of DAPI staining showed no evident difference either, except that in the
`aminoalkylsilane-treated glass preparations the background is also cleaner than that in
`ethanol/ether-cleaned slides.
`
`Metaphase spreading
`The spreading of metaphases prepared from blood cell cultures of three healthy donors
`and three haematology patients (one bone marrow and two lymphocyte cultures) was
`measured and compared for preparations on aminoalkylsilane-treated glass slides and
`glass slides cleaned with ether/ethanol. The results are shown in Table 1. Except for the
`metaphases of the bone marrow culture (D),
`the mean metaphase areas on all
`
`Page 7 of 12
`Page 7 of 12
`
`

`
`340
`
`VAN PROOIJEN-KNEGTETAL.
`
`Table 1. Average areas of Giemsa-stained metaphases (expressed in pixel points
`: s.E.M.) determined with a TAS in three areas of 10.5 mm2 in each of four prepa-
`rations on aminoall<ylsilane—treated glass slides and on routinely cleaned slides.
`
`Aminoalkylsilane-treated glass
`
`Alcohol/ether cleaned glass
`
`Healthy donors A
`B
`C
`
`Patients
`
`D*
`E
`F
`
`* B0116 marrow culture.
`
`639 (i12.'l)
`629 (13.8)
`689 (111.7)
`
`395 (i5.9)
`702 ($16.8)
`631 (:15.8)
`
`564 (i3.9)
`581 (i1.7)
`58] (i9.9)
`
`440 (i 6.6)
`554 (113.8)
`538 (111.8)
`
`aminoalkylsilane-treated glass preparations were larger than those on normal glass
`slides. Less cells were counted in the central parts of the aminoalkylsilane-treated glass
`preparations than in routine preparations, but microscopical inspection showed that in
`the latter preparations, the cells had spread more homogeneously all over the glass
`slides. The ratio between the number of metaphases and the number of nuclei per area
`was found to be the same for both types of preparations.
`
`Quimzcrine staining intensity
`Visual comparison of the staining results obtained with the equilibrium method, using
`the low concentration of quinacrine (0. 002%), after different staining times, showed that
`sufficient contrast and banding for Visual observation was obtained after about 90 min.
`for the experiments described hereafter, equilibrium staining was always performed for
`120 min. The fluorescence emission intensity of the equilibrium-stained preparations
`seemed Visually to be slightly less than that of optimally stained ‘routine’ preparations.
`The staining intensities of routinely and equilibrium-stained preparations were then
`compared by determining the exposure times necessary to obtain fluorescence
`microphotographic negatives with the same optical densities. The results of this
`experiment show that equilibrium-stained preparations have at least the same emission
`intensity as the preparations stained with the 0.1% medium. Under the illumination
`conditions applied, using the High Contrast Copy Film these exposure times were 10 s
`for brightly fluorescent metaphases in preparations stained with the 0.1% quinacrine
`medium, and 6-7 s for equilibrium-stained (0.002%) metaphases. With the more
`sensitive Technical Pan Film, a 2-5 s actual illumination time is necessary to obtain
`microphotographs with sufficient contrast (Fig. 4).
`
`I-"eu1gen—DNA absorbance values
`To check a possible influence of the aminoalkylsilane coating on the Feulgen stainability,
`the absorbance values of chicken erythrocyte nuclei stained in preparations on both
`types of glass slides were measured. The mean integrated A550 per 0.25 psmz of 60 nuclei
`(ten in each of three areas in two slides) per type of preparation were measured.
`
`Page 8 of 12
`Page 8 of 12
`
`

`
`Metaphase chromosomes on aminoalkylsilane-treated slides
`
`341
`
`‘:3
`C’.
`
`1.0
`
`2
`
`5
`
`10
`
`20 sec illum.
`
`Fig. 4. The relations between integrated corrected optical density values in microfluorographic
`negatives on Technical Pan Film of quinacrine-stained chromosomes no.2, and the actual
`exposure time. Each point is the mean of six measurements (vertical bars = s.D.).
`
`The means .iS.E.M. were:
`
`:
`alcohol/ether—cleaned glass
`aminoalkylsilane-treated glass:
`
`26.45 i 0.13
`26.41 i 0.15.
`
`The mean Feulgen—DNA absorbance (is.E.M.) of human no. 2 chromosomes measured
`as the optical density per 1/64 #1112 in photographic negatives were for preparations on:
`
`:
`alcohol/ether—cleaned glass
`aminoalkylsilane-treated glass:
`
`40.79 i 0.45
`40.77 i 0.45.
`
`To check whether the difference in staining procedure influenced the intensity of
`Feulgen staining (carried out after quinacrine staining),
`the integrated Feulgen
`absorbances were determined for no. 2 chromosomes from photographic negatives.
`The results are shown in Table 2.
`
`Table 2. Integrated optical density value sof human metaphase
`chromosomes no. 2 after Feulgen—p‘ararosaniline staining
`measured on photographic negatives. Each value repre sents the
`mean of 20 chromosomes (10 in each of two preparations).
`
`Preceding treatment
`
`A —
`B quinacrine 0.1%
`C quinacrine 0.002%
`
`Integrated optical density
`per I/64 pm? (:s_E.M.)
`
`408 (4.5)
`414 (4.5)
`409 (14.3)
`
`Page 9 of 12
`Page 9 of 12
`
`

`
`34?.
`
`Discussion
`
`VAN PROOI]EN—KNEGTETAL.
`
`followed by chemical
`Treatment of porous glass beads with aminoalkylsilane,
`conversions, has enabled RNA and heat—denatured DNA to be attached for affinity
`chromatographic purification of nucleic acid binding proteins (Robinson 62.‘ 111., 1971). In
`the present study, glass slides were treated with 3~aminopropyltriethoxysilane to
`reduce or prevent non-specific binding of basic dyes to the glass surface. This prin-
`ciple worked in practice
`as can be
`concluded from the
`reported results.
`Aminoalkylsilane-modified glass slides also were found to decrease the non-specific
`background staining when treated with a fluorochrome-labelled antiserum (Van
`Prooijen et £21., unpublished results). An unexpected effect of the arninoalkylsilane
`treatment of glass which was observed, is that metaphases spread better on such slides
`than on ethanol/ether-cleaned glass slides.
`As a result of the special culture and preparation procedure applied in our laboratory,
`metaphase chromosomes have already been relatively well-spread on normal glass
`slides (Fig. 2b). However,
`the average area of metaphases in preparations on
`aminoalkylsilane-treated glass is 11% larger. In agreement with this larger area, fewer
`overlapping chromosomes are found in these metaphases. In our preparations the
`average number of overlaps per metaphase is two per metaphase spread on routinely
`cleaned glass slides and one on aminoalkylsilane-treated glass slides. Furthermore, the
`distribution of
`the cells over
`the aminoalkylsilane-treated glass
`slides is more
`homogeneous over the total area than in preparations on normal glass where they
`sometimes tend to remain closely around the position where the drop with cell
`suspension hit the microscope slide.
`_
`The reproducibility and the constancy of the quinacrine staining results were further
`improved by the development of a staining method that does not require a washing
`procedure to remove any excess of dye. A medium containing a very low concentration
`(0.002%) of quinacrine allows optimal Staining results to be obtained within 90 min and
`can also be used as an embedding medium. In this way slides were prepared showing
`high-quality banding patterns in all well-spread metaphases. This equilibrium staining
`method works both on routinely cleaned and on aminoalkylsilane-treated glass slide
`preparations. The staining intensity obtained in these preparations after 30 min visually
`seemed to be slightly less than in the best-stained metaphases when using the 0.1%
`medium. This phenomenon most probably can be ascribed to the (weakly) fluorescent
`background resulting from the quinacrine-containing embedding medium. The
`exposure times necessary to produce microphotographic negatives of a defined contrast
`are shorter for equilibrium-stained preparations (120 min) than for preparations stained
`with the 0.1% medium (Fig. 4).
`Neither the aminoalkylsilane treatment of the glass slides nor the equilibrium staining
`influence the photodecomposition of DNA which occurs during the observation of
`quinacrine-stained chromosome preparations in the fluorescence microscope. During
`observation or microphotography of quinacrine-stained chromosomes preparations (for
`reasons of pre—identification) a decrease of Feulgen stainability can result, as was
`
`Page 10 of 12
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`
`

`
`Metaphase chromosomes on aminoal1<ylsilane~treated slides
`
`343
`
`(1977). It can be concluded from their results that this
`described by Bosman et al.
`decrease is related to the strength and duration of irradiation and to the amount of stain
`bound to the chromosomes.
`
`Our results also show that the decrease in Feulgen stainability for both types of glass
`and independent of the staining procedure is, at least partially,_ correlated with the
`brightness of the fluorescence emission. During prolonged irradiation,
`the Q—band
`regions lose more Feulgen stainability than the interbands, which can result in a
`detectable ‘reverse Feulgen—banding’ pattern.
`In conclusion, the use of aminoall<ylsilane—treated microscope glass slides has several
`advantages. Their preparation is relatively easy; spreading of metaphases on the glass
`surface is improved, leading to less overlap of chromosomes; non-specific background
`staining is decreased not only for quinacrine—staining, but also for Giemsa and DAPI
`staining. Another step towards a more consistent production of high quality
`chromosome banding has been made by the development of an equilibrium
`quinacrine—staining method. Both procedures
`contribute
`to
`the quality and
`reproducibility of chromosome staining results and in this way to a more optimal visual
`or automated chromosome analysis.
`
`Acknowledgement
`
`This work was supported by ’Het Praeventiefonds’, ‘s-Gravenhage, Grant Nr. 28-394.
`
`References
`
`(1977) Influence of Q- and
`BOSMAN, F. r., VAN DER PLOEG, M. & GERAEDTS, 1. P. M.
`G—banding on the Feulgen—stainability of human metaphase chromosomes. Histochem. I. 9,
`31-42.
`
`BOSMAN, F. TE-1., VAN DER PLOEG, M., SCHABERG, A. at VAN DUIIN. P. (1975) Chromosome
`
`preparations of human blood lymphocytes - evaluation of techniques. Geneticzz 45, 425-33.
`BOSMAN, F. T., VAN DER PLOEG, M., VAN DUIJN, P. & SCHABERG, A. (1977) Photometric
`
`determination of the DNA distribution in the 24 human chromosomes. Expl Cell Res. 105,
`301-11.
`
`DUUNDAM, W. A. L. & VAN DUIJN, P. (1973) The dependence of the absorbance of the final
`chromophore formed in the Feulgen—Schiff reaction on the pH of the medium. Histochemie 35,
`373-5.
`
`GRAU MANN, W. (1952-3) Zur Standardisierung des Schiffschen Reagens. Z. wiss. Mikrosk. 61,
`225-6.
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`ROBINSON, P. 1., DUNNILL, P. & LILLY, M. D. (1971) Porous glass as a solid support for
`immobilisation for affinity chromatography of enzymes. Biochim. biophys. Acta 242, 659-661.
`SERRA, J. (1974) Theoretical bases of the Leitz-Texture—Ana1ysing-System. Leitz Mitt. Suppl. 1,
`125-36.
`
`SUMNER, A. T., EVANS, H. 1. & BUCKLAND R. A. (1971) New technique for distinguishing
`between human chromosomes. Nature New Biol. 232, 31-2.
`(1974a) High—reso1ution
`s.
`VAN DER PLOEG, M., VAN DUIIN,
`P.
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`Page 12 of 12