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Preparation of stock solutions for FISH

note: use for all preparations deionised, bidestilled and sterilized water

RNase 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 100C
-cool slowly to room temperature (RT)
-make aliquots of 100 l
-store at -20C
-keep 1 tube for direct use at 4C

500 ml 8% Paraformaldehyde pH 7
-take 40 g of paraformaldehyde and dissolve in 450 ml of water
-put in WWB of 40C
-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 4C

Deionized formamide
-mix 50 ml formamide and 5 g ion exchanger (Amberlite, Sigma)
-stirr for 2 hrs at RT
-filter through Whatman filter n1
-store at 4C

Biotinylated goat anti-avidin (0.5mg/ml)
-dissolve 0.5 mg in 1 ml of water
-make aliquots of 100 l
-store at -20C

Neutralite Avidin FITC
-dissolve the lyofilized material in 1 ml of 1xPBS
-store at 4C

Mounting 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 l and store at -20C
-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 70C, 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 l PI stock (1 mg/ml)
-mix very well
-store in tubes of 1 ml at -20C

50% formamide / 12.5 % dextranesulphate / 2xSSC
-dissolve 12.5 g dextranesulphate in 100 ml 50% deionized formamide / 2xSSC
-shake for 3 hrs at 70C (WWB)
-make aliquots of 1 ml
-store at +4C

0.5 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)

-dissolve 175.3 g NaCl and 88.23 g tri-sodium-citrate in 900 ml of water
-put to pH 7.0 with HCl

-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 ml

0.5 M MgCl2
-dissolve 50.82 g MgCl2.6H2O in 500 ml water

3 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 4C

60% 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 HCl

1.0 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 HCl

0.5 % blocking reagent
-dissolve 0.05 g blocking reagent in 10 ml of 0.1 M tris + 0.15 M NaCl
-put in WWB of 70C to dissolve completely
-store at -20C

Mounting medium with DAPI
-mix 1 ml of Vectashield (Vector) with 10 l of DAPI (10 ng/l)

10 % pepsin
-dissolve 10 g pepsin in 100 ml water
-make aliquots of 100 l and store at -20C