9. Antifade solution
100mg p-phenylenediamine dihydrochloride in 10mL PBS. Adjust to pH 8.0 with 0.5M carbonate-bicarbonate buffer (0.42g NaHCO3 in 10mL dH2O, adjust pH to 9.0 with NaOH). Add to 90mL with glycerol. Filter with 0.22m membrane to remove undissolved particals. If necessary, add 0.5-1mg/mL DAPI.
OR: DAPI-solution: Dissolve 5 mL of DAPI (4,6-diamidino-2-phenylindol.2HCl stock- solution; Serva) in 100 mL 4X SSC/0.2% Tween; make fresh as required.
OR: 70% (v/v) deionized formamide, 10% (v/v) filtered double-distilled water, 10% (v/v) 20X SSC, 10% (v/v) phosphate buffer, make fresh as required.
2. Procedures for Fluorescence In Situ Hybridization (FISH):
Pre-treatment of slides
1.(Optional) If the slide is not dry enough, dehydrate the slide by immersing the slide into 100% ethanol for 1 min. Air dry.
2.Pretreat slide with 200mL diluted RNase solution (0.1mg/mL) for about 30 to 60 minutes at 37℃.
3.Wash slide with 2X SSC for 5 minutes (with agitation).
4.Pepsin treatment: (Optional, if the chromosome targets are bone marrow smear, bone marrow progenitors from methyl cellulose-grown colony assays, tumour preparations)
i.Add 200mL diluted (with 0.01M HCl) pepsin to the slide
ii.Incubate slides at 37℃ for 5-10 minutes.
iii.Wash 2X with 1X PBS for 5 min at RT with shaking.
Note: over-digestion can also cause problems (loss of cells from the slide), so only use when absolutely necessary.
5.Place slides in PBS/50mM MgCl2 for 5 min.
6.(Optional) Fix in PBS/50mM MgCl2/1% formaldehyde for 10 min.
7.Wash in PBS for 5 min (with agitation).
8.Dehydrate the slide with 70%, 90%, and 100% ethanol for 1-2 min each and allow to air dry. Slides can be stored desiccated at 4℃ for up to one month before use.
i.Add 2mL (100ng) diluted Biotin-labeled DNA + 1mL (2.5mg) Cot-1 DNA + 7mL MM2.1(warmed to RT).
ii.Denature the hybridization mix at 75℃ for 5-7 minutes.
iii.Place the probe on ice for 3-5 mins. (very IMPORTANT!)
iv.Transfer the denatured probe to 37℃ for 15 min – 2 h for prehybridization.
