FISH on Schistosoma mansoni metaphases

Prepare metaphases from intramolluscan stages of Schistosoma mansoni

The protocol is based on H. Hirai and P.T. LoVerde Parasitology Today, vol. 11, no. 8. 1995

Prepare probes for repetitive DNA

Prepare biotinylated probes using the BioPrime DNA labeling system (invitrogen #18094-011). We use PCR products cloned into plasmids. There is no need to remove the vector, the plasmids can be used directly.

Do FISH

Day 1:

RNAse treatment

for 1 ml:

70 l on each slide, cover with 1x2 cm parafilm, incubate in a humid chamber at 37C for 1 hour

Pepsin treatment

for 200 ml:

incubate in a joplin jar 30 min at 37C

Dehydration

Denaturation

Slides:

for 200 ml pH 12.5:

incubate in a joplin jar 4:30 min at RT

dehydrate immediately:

Probe:

for 10 slides prepare

21 l of this + 4 l of biotinylated probe

denature at 99C for 5 min, put in ice immediately, leave on ice for 5 min

add 25 l on each slide, cover with a piece of parafilm

incubate over-night at 37C in a humid chamber

Day 2:

prepare TBST (20 ml 1 M Tris/Cl pH7.5, 30 ml filtered 5M NaCl, 0.5 ml Tween 20, to 1 l with distilled water)

1 wash in 40% formamide, 2xSSC at 42C, 20 min (temperature inside the jar!)

1 wash in 2xSSC at 42C, 20 min

1 wash in 2xSSC at RT, 20 min

Blocking in TBST, 5% milk in the jar at RT for 30 min

add 100 l of a 1:100 dilution of Streptavidine-FITC (invitrogen #43-4311) (10 l in 1 ml TBST 5% milk) on each slide

cover with parafilm

incubate in a humid chamber at 37C for 1 hour

wash twice for 10 min at RT in 200 ml TBST on a shaker

embed in 10 l vectashield with 5g/ml propidiumiodide

keep at 4C until observation




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