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Fig 1 (FISH guide)
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Legend Fig.1. a) Chemical aging setting, using the metal block of a thermocycler: slides are placed on the metal block, 150-200ul ethanol pipetted onto them and covered with coverslip. A plastic cover, containing gauze or paper soaked in ethanol is placed so as to cover the slides and maintain an ethanol-saturated atmosphere during the heating cycle. b-f) Non-aged slides. Slides were kept 1-2 hours at RT, then subjected to pretreatment in: pepsin (c,b), 2xSSC (d) and trypsin (e,f). A BIO-labeled commercial chromosome 1 centromere was hybridized onto slides for 30 minutes, then detected with avidin-FITC. g-l) Aged slides: pepsin vs. trypsin pretreatment. Same chromosome 1 centromere was used in all six images. (g,h) Examples of pepsin pretreatment onto non-aged (g) and chemically aged (h) slides; (i,j) trypsin pretreatment on non-aged (i) and chemically aged (j) slides; k,l) nuclei hybridizations on chemically aged slides pretreated with pepsin (k) and trypsin (l). Fig 1 results indicate that, chemically aged chromosomes and nuclei preserve better their architecture compared to non-aged slides, without losing in hybridization quality. At the enzyme concentrations used (see text), pepsin pretreatment preserves better the architecture of nuclei and chromosomes than trypsin. Hybridization was more efficient on pretreated compared to non-pretreated slides. All posthybridization washes were done at 42 C.
