PREPARATION OF STOCK SOLUTIONS FOR FISH

PREPARATION OF STOCK SOLUTIONS FOR FISH note: use for all preparations deionised, bidestilled and sterilized waterRNase A (10 mg/ml)-dissolve 10 mg in 1ml of 10 mM Tris-HCl (pH 7.5) and 15 mM NaCl-put this solution in a corning tube in warm water bath (WWB) for 15 min at 100°C-cool slowly to room temperature (RT)-make aliquots of 100 µl-store at -20°C-keep 1 tube for direct use at 4°C500 ml 8% Paraformaldehyde pH 7-take 40 g of paraformaldehyde and dissolve in 450 ml of water-put in WWB of 40°C-add drops of 5 N NaOH, and mix once in a while-continue by adding NaOH till everything is dissolved-kool down (RT)-bring solution to pH 7 with 6 N HCl-add water to 500 ml-filter through S&S filter-store at 4°CDeionized formamide-mix 50 ml formamide and 5 g ion exchanger (Amberlite, Sigma)-stirr for 2 hrs at RT-filter through Whatman filter n°1-store at 4°CBiotinylated goat anti-avidin (0.5mg/ml)-dissolve 0.5 mg in 1 ml of water-make aliquots of 100 µl-store at -20°CNeutralite Avidin FITC-dissolve the lyofilized material in 1 ml of 1xPBS-store at 4°CMounting medium for slides-dissolve 2.42 g Tris and 0.02 g NaN3 in 100ml water-bring to pH 7.5 with HCl, this results in solution of 0.2 M Tris-HCl and 0.02 % NaN3-dissolve 2 mg of propidiumiodide (PI) in 2 ml of the above mentioned Tris-buffer, this gives a PI stock of 1 mg/ml-make aliquots of this PI stock of 10 µl and store at -20°C-put 0.69 g of di-azo-bicyclo-octane (DABCO) in 50 ml Falcon tube-add 30 ml of 100% glycerol-dissolve in WWB of 70°C, solution of 2.3 % DABCO in glycerol-put 9 ml 2.3 % DABCO in glycerol together with 1 ml 0.2 M tris-HCl 0.02 % NaN3 5 µl PI stock (1 mg/ml)-mix very well-store in tubes of 1 ml at -20°C50% formamide / 12.5 % dextranesulphate / 2xSSC-dissolve 12.5 g dextranesulphate in 100 ml 50% deionized formamide / 2xSSC-shake for 3 hrs at 70°C (WWB)-make aliquots of 1 ml-store at +4°C0.5 M Phosphate buffer pH7.0-for 100 ml prepare separate 100 ml of 6.8045 g KH2PO4 (1) 100 ml of 7.098 g Na2HPO4(2)-take 77 ml of solution (2) and mix with 33 ml of solution (1)20xSSC-dissolve 175.3 g NaCl and 88.23 g tri-sodium-citrate in 900 ml of water-put to pH 7.0 with HCl10xPBS-dissolve 16.02 g Na2HPO4.2H2O and 73.84 g NaCl in 900 ml water (solution 1)-dissolve 2.76 g NaH2PO4.H2O and 16.36 g NaCl in 200 ml water (solution 2)-bring solution 1 to pH 7.0 with solution 2-add water to 1000 ml0.5 M MgCl2-dissolve 50.82 g MgCl2.6H2O in 500 ml water3 M Naacetate pH5.6-dissolve 24.61 g Na-acetate in 90 ml water-bring to pH 5.6 with acetic acid-put to 100 ml with water-store at 4°C60% formamide/2xSSCP (moist chamber)-dissolve 17.53 g NaCl, 8.82 g tri-Na-citrate and 5.68 g Na2HPO4 in 200 ml water (10xSSCP)-mix 300 ml formamide with 100 ml 10xSSCP and 100 ml water, bring to pH 7.0 with HCl1.0 M Tris + 1.5 M NaCl pH 7.5-for 5 l take 605.7 g Tris and 438.3 g NaCl, dissolve in 4.5 l water-bring to pH 7.5 with 12 N HCl0.5 % blocking reagent-dissolve 0.05 g blocking reagent in 10 ml of 0.1 M tris + 0.15 M NaCl-put in WWB of 70°C to dissolve completely-store at -20°CMounting medium with DAPI-mix 1 ml of Vectashield (Vector) with 10 µl of DAPI (10 ng/µl)10 % pepsin-dissolve 10 g pepsin in 100 ml water-make aliquots of 100 µl and store at -20°C