There are three variables to be considered during the posthybridization washes: the composition of solutions, washing temperature and the washing time.
After the coverslip is carefully removed using thin-tip tweezers, the slide is passed through the following washes:
• first wash, in 50%FA/2xSSC. At this step, both the excess probe and the hybridization buffer are removed from the slide. Some laboratories allow the coverslip to "fall off" the slide at this step. This wash is usually done in a Coplin jar at temperatures from 37-45° C, three times 5 minute each. Higher temperatures (up to 65 C) can be used for short periods of time as well, but run the risk of losing too much of the hybridized probe DNA.
Note: if the SSC concentration is lower than 2x, because of the high FA concentration in the buffer, there will be a significant loss of hybridization signal.
• second wash (the stringent wash), in 0.1-2x SSC, usually three times 5 minutes each. This step is used primarily to remove the non-specific and/or repetitive DNA hybridized to the cells and chromosomes. The less concentrated the salt solution and the longer the duration of the wash and the temperature, the higher the stringency and the more DNA will be removed. This wash can be done at temperatures between 25 and 75 C, in two-three steps, 2-5 minutes each. It is impossible to present a standard procedure, but there are some guidelines that can help. For very short DNA probes (0.5-3 kb) or very complex probes (chromosome paint probes, CGH), the washing temperature should be lower (up to 45 C) and the stringency lower as well (1x-2x SSC). When using single-locus, large probes (cosmids, BACs, P1s, YACs), the temperature should be around 65 C and the stringency high (below 0.5xSSC). The temperature and stringency should be highest for repetitive probes (such as alpha-satellite repeats).
• post-antibody washes. After each 10 minute antibody incubation, the excess antibody needs to be rinsed off, before another layer of antibody is added. This wash is usually performed in a 4x SSC/0.1% Tween solution, at 25-45° C, in three steps, 2-5 minutes each.
• salt removal: before the antifade solution is added, the slide should be rinsed a few seconds in water to remove the SSC, and then air-dried at room temperature.
If desired, the washing protocol can be stopped after the second wash and the slide can be rinsed briefly in water, air dried, and then stored in a refrigerator or even at room temperature for a few days, before antibody detection is performed.
We compared three commercially available antifade solutions with a self-made antifade mounting medium, prepared according to a published protocol (made by dissolving 0.233g DABCO in 800 ?l water, then adding 200 ?l 1M Tris pH 8.0 and 9 ml glycerol solution) (Current Protocols In Human Genetics; 4.3.17). In our experience, there was no visible difference between the DABCO mounting medium and commercial antifade solutions. The DABCO medium is very inexpensive and allows visualization of fluors across the entire spectrum (DAPI, AMCA, FITC, R6G, Cy2, Cy3, Rhodamine, Texas Red, Cy3.5, Cy5, Cy5.5, Cy7). The solution should be stored in small aliquots (for 20-30 slides) at -20° C or -70° C. The vials should be kept as little as possible at room temperature (enough to get the required amount for the slides) and should be stored back in the freezer. It is very important that the antifade is placed on air-dried slides, without being diluted by water or buffer residues on the slide. Therefore, after the last posthybridization wash (in 4xSSC/Tween or similar solution) all slides should be rinsed for 3-5 seconds in distilled water (to wash away the salt) and air-dried at room temperature. 20 ?l antifade are placed on a slide and are covered with a 24x50 coverslip. The edges of the coverslip can be sealed with nail polish, however, this step is not necessary. Slides can be stored 2-3 weeks at 4 C or longer at —20° C. If slides are kept at room temperature and the antifade oxidizes, slides can be rinsed in washing solution (4xSSC/Tween), and a new batch of antifade can be used on the slide. The nail polish may increase the shelf-life of FISH slides, as it decreases antifade oxidation by blocking air exposure.
As described for CCK, if necessary, the antifade/mounting medium can be rinsed off the slides and a new layer of antibodies can be added.
DAPI or PI counterstaining.
Counterstaining reagents such as DAPI (blue) or Propidium iodide (PI, orange-red) can be used to stain slides either separate (in a jar, diluted in buffer) or directly mixed with the antifade solution. Although both methods work, the quality of the banding pattern (DAPI) and the overall quality of the staining (contrast, brightness) is higher when the counterstain is added separately from the antifade. This is especially important for the CGH protocol, in which a good DAPI banding pattern is essential to identify the chromosomes. As general guidelines, DAPI and propidium iodide staining solutions are used at a concentration of 200 ng/ml DAPI or PI in 2xSSC or PBS.
The jar with staining solution is wrapped in aluminum foil or is taped with black tape (to protect it from light) and can be used repeatedly, for up to 2-3 months if stored at 4° C. Slides are incubated 2-10 minutes in the staining solution and are rinsed 5-10 minutes in 2xSSC or PBS afterwards (to wash off excess dye). Then, the slides are rinsed for a few seconds in water to remove any salt traces, then air dried at room temperature and mounted with antifade solution.
When DAPI or PI are used within the antifade solution, the usual concentration is about 1ng dye/ml antifade.
DAPI bandingcan be improved by:
The intensity of counterstaining the chromosomes should also be adjusted according to the equipment used to analyze the slides. If, for example, the fluorescent microscope is equipped with a wider bandpass FITC filter, a very bright PI counterstain of the chromosomes will become visible when the slide is examined through the FITC filter. In this case, image capturing of FITC probes using common, non-color, CCD cameras will be difficult. In such cases, intensity of counterstaining should be decreased.
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Last modified on: Feb12, 2001