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Protease pretreatment
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Type of proteases
Many tests performed in this laboratory showed that protease pretreated slides ALWAYS worked better for FISH, CGH or M-FISH, when compared with non-pretreated slides.
The most common enzyme used was pepsin. Although both pepsin and trypsin can be used, pepsin has the advantage that it can be easily inactivated by pH changes, and the reaction is easier to control. A stock solution of 10% pepsin in water (or 50% glycerol) can be prepared at one time, aliquoted and stored years at -20° C. The working solution was 0.005% enzyme in 0.01N HCl.. Incubation time is 1 minute for chemically aged slides, and 10-15 minutes for slides aged overnight at 65° C, and is followed by a few seconds incubation in PBS and ethanol series. Trypsin can also be used at 0.5-1mg/ml in isotonic buffer, similar to the banding protocol. After 3-10 seconds incubation, enzyme activity is stopped by immersion in 0.5-1% FCS for a few seconds. The slide is then rinsed in PBS (2-3 minutes) and in an ethanol series. Trypsin concentration was about ten times higher than pepsin concentration, thus requiring much shorter digestion times.
A prescribed, fixed amount of time for enzyme pretreatment of slides is difficult to give. Pretreatment depends on the tissue of origin, the quality of the pellet (primarily influenced by the length of colcemide exposure and length of hypotonic treatment) and purpose of hybridization. For example, in our laboratory, fibroblasts or tumor cells required less pepsin pretreatment than lymphocytes. A cell suspension with a lot of cytoplasmic residue, does not spread well and requires longer protease pretreatment compared to better quality cell material. Slides for CGH or M-FISH, where a good chromosomal architecture is important (for DAPI banding), require somewhat less protease pretreatment. For FISH with cosmids or plasmids, the protease pretreatment can be increased, as good hybridization signals are more important than a perfect chromosome architecture. For FISH with small probes (1-2kb) pretreatment is even more important, as it makes proteic structures more permeable and increases the chance of targeting the desired DNA sequence.
Protease pretreatment before or after denaturing ?
Slides were treated with pepsin or trypsin before or after denaturing, and FISH results compared. Either approach resulted in improved FISH signals.
- Protease digestion before denaturing
a). Using non-aged slides
Slides kept only 1-2 hours at room temperature were pretreated with 0.005% pepsin for 1 minute, 0.5mg/ml trypsin for 10 seconds or just kept in 2x SSC solution for 20 minutes prior to denaturing. Chromosome architecture was better preserved when using pepsin (compare Fig.1b and 1e). The use of trypsin (either before or after denaturing) resulted in "puffy" chromosomes and did not allow reasonable DAPI banding. Nuclear structure was also better preserved when using pepsin over trypsin or SSC (compare Fig. 1c, 1d and 1f). Hybridization signals were weak when only SSC was used (Fig. 1d) but were strong when a protease digestion was performed (Fig. 1c and 1f). After trypsin pretreatment, nuclei acquired a "circumference" of stronger DAPI staining, probably due to over-digestion of the cellular material and partial loss of DNA after denaturing. After pepsin digestion, nuclei showed more uniform DAPI staining.
b). Using dry-heat aged slides
In Fig. 2a-c, slides were aged 2 hours at 37 C dry heat, then were subjected to 15 seconds (2a), 6 seconds (2b) or no trypsin pretreatment (2c), and then were denatured and DAPI stained. Trypsin treatment proved too extensive, and the only acceptable slide was the one without exposure to the protease.
c) Using chemically aged slides (ethanol)
In a similar experiment (Fig. 2d-2f), slides were aged 2 minutes in ethanol, then either used directly (2d), or pretreated in trypsin for 10 seconds (2e) or 5 seconds (2f). Chemical aging preserved chromosome morphology on all three slides, whereas protease pretreatment improved FISH signals (compare Fig. 2d with 2e).
- Protease digestion after denaturing
A few hybridization tests were done in which slides were first aged and denatured and only afterwards subjected to enzymatic pretreatment. Although protease pretreatment improved FISH results, chromosome morphology was not as good.
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