Chromosomes counterstaining

Some fluorescent DNA stains can also be used for chromosome counterstaining, for detection of hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization assays or for identifying apoptotic cells in cell populations (http //probes.invitrogen.com/handbook/sections/0806.html). For instance, Vybrant Apoptosis Assay Kit 4 (Molecular Probes) detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide. 

SYTOX blue nucleic acid cell stain S11348 Molecular probes Xex 470 nm 480 nm SYTOX blue nucleic acid stain is an excellent blue-fluorescent nuclear and chromosome counterstain that is impameant to live cells, making it a useful indicator of dead cells within a population. 

Light micrograph of late mitosis in a plant. Microtubules are stained red and chromosomes are counterstained blue. (Courtesy of Andrew Bajer.)... 

Add 60 xL of DAB substrate solution and leave for 5 min at room temperature. Rinse the slides in tap water to stop the reaction (j Note 10). Counterstain for 5 min in 10% Giemsa, rinse in buffer, and air-dry (se Note 11). Permanent mounting without subsequent foding of the chromosome and/or hybridization signal is best achieved by mounting slides in Xam. 

Chromosomes will appear fuzzy and faintly counterstained, nuclei will appear ghost-like, and central areas of DNA may even be removed from nuclei. Under-denaturation will lead to poor signal strength, with strong counterstaining of well-defined chromosomes and nuclei. From this point of view, formalin-fixed, paraffin-embedded tissue is the hardest to work with. Table 4 shows some of the parameters we have used on a series of breast samples. 

Fig. 18. Metaphase image of a mouse/human hybrid bearing multiple human chromosomes, probed with FITC-biotinylated human DNA (light gray) and counterstained with propidium iodide (dark gray). The human chromosomes show up in light gray as a result ofacombined signal of FITC and PI. (Sample courtesy of Dr. Roger A. Schultz, University of Maryland School of Medicine, Baltimore, MD.)...

Fig. 22. Male peripheral blood mononuclear cells labeled with biotin- l chromosome probe/avidin (F1TC green) and counterstained with propidium iodide (red). Seventeen optical sections (27 x 27/zm each) were imaged at 0.2

Figure 7. Occurence of chromatid breaks in centromeric r on 7 h after 2 Gy irradiation. Metaphasespreads of bone marrow cells from irradiated PARP-2 (panel A) and PARP-2 mice (panel B-Q. Arrows point to absence of centromeric signals. Giemsa staining (A,B) Chromosome painting with a centromeric probe, counterstaining with DAPI (C). (Taken from M6iissier-de Murcia et al, with permission). A color version of this figure is available online at www.Eurekah.com.

Chromosomal localization of the axl gene is done by fluorescence in situ hybridization (FISH). The biotin-labeled axl probe is from a cosmid vector containing a 41-kb genomic fragment. The slides with human metaphase cells for chromosomal DNA to be denatured are immersed into a solution of 70 % formamide-x4 in saline sodium citrate buffer (SSC) atpH 7.0 (0.15 M NaCl 0.015 M sodium citrate) at 70 °C for 2 min. For the slides, the hybridization mixture kept at 75 °C consists of 50 %formamide,xl SSC, 10 % dextran sulfate pH 7.0, and 150 mL unlabeled sonicated total human genomic DNA add 50-100 ng labeled probe. The mixture is incubated at 37 °C for 10 min. The slides are washed in 50 % formamide and SSC x3, 5 min each at 40 °C and in SSC x3 at 40 °C. The detection reagent consists of x4 SSC-0.1 % Triton, and 1 % bovine semm albumin for 3 washes 3 min each at 40 °C. The slides are incubated with fluorescein isothiocyanate-conjugated avidin at 37 °C for 30 min. Metaphase cells are counterstained with 4,6-diamidino-2-phenylindole dihydrochloride 200 ng/mL in 2x SSC for 5 min at... 

Schweizer, D. (1980) Counterstain-enhanced chromosomes banding Hum. Genet. 57,1-14. 

Examine chromosome preparations to determine whether to counterstain since counterstaining can obscure weak in situ hybridization signal. 

Chromosomes with HRP signals should be counterstained with Giemsa as described below. 

Chromosomes with fluorescent signals can be counterstained with Hoechst 33258 as described below. However, banding patterns produced by the fluorescent DNA stains are often difficult to compare with the published polytene chromosome-banding patterns. We find it more convenient to simply use phase contrast to correlate the fluorescent signal with the corresponding chromosome bands. 

The general strategy for FISH is to equilibrate the tissue in buffered formamide, to add the probe(s), and to denature both the chromosomal DNA and probe together by heat treatment. The probe is then allowed to anneal for several hours at an appropriate temperature, unbound probe is washed away, and secondary detection (if required) is performed. The nuclear DNA is then usually counterstained, and the sample is mounted for microscopy. For experiments in which immunolocalization of other cellular components is desired, these staining steps can conveniently be performed after hybridization. 

---