FISH on Schistosoma mansoni metaphases

Prepare metaphases from intramolluscan stages of Schistosoma mansoni

• prepare 50 ml of fixative (ethanol:acetic acid = 3:1)
• dissect 2-3 snails 28-29 days post infection (time might depend on strain) and prepare sporocysts, remove other tissues if possible
• treat the sporocysts with a 1:10 dilution in distilled water of Demecolcine in Hank's balanced salt solution (HBSS) (Sigma D1925), final concentration 1 µg/ml for 20 min at room-temperature (RT). Demecolcine is less toxic than colchicine.
• transfer 1 ml of fixative to the sporocysts in Demecolcine and tease the tissue with needles into small pieces
• transfer into 10 ml in a 15 ml Falcon tube
• triturate the suspension 5 times with a 5 ml syringe and a 18 gauge needle
• leave the tube at room temperature for 10 min to allow large tissue to settle and transfer only the supernatant to a new 15 ml Falcon tube
• centrifuge the new tube at 1500 rpm for 5 min at 10°C, remove supernatant by inversion
• resuspend in fresh fixative
• repeat centrifugation
• make cell suspension using 200-300 µl of fixative
• apply 1 drop of the suspension onto a clean glass slide
• place the slide on a horizontal surface and add 1 drop of fixative on the preparation just before the fixative dries
• repeat this step
• let dry and observe under a phase contrast microscop
• you should observe at least 2 metaphases per slide
• store the slides at 4°C in a closed container

Prepare probes for repetitive DNA

Do FISH

Day 1:

RNAse treatment

for 1 ml:

• 900 µl sterile destilled water
• 100 µl 20x SSC
• 3 µl 30 g/l RNAse (Sigma R4642)

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

Pepsin treatment

for 200 ml:

• 192 ml distilled water
• 8 ml 0.25 N HCl (97.5 ml distilled water + 2.4 ml 37% HCl)
• 100 µl 10% pepsin

incubate in a joplin jar 30 min at 37°C

Dehydration

• 5 min at RT in 70% ethanol
• 5 min at RT in 95% ethanol
• air dry completely

Denaturation

Slides:

for 200 ml pH 12.5:

• 177 ml distilled water
• 20 ml 20x SSC
• 3.4 ml 3M NaOH

incubate in a joplin jar 4:30 min at RT

dehydrate immediately:

• 5 min at RT in 70% ethanol
• 5 min at RT in 95% ethanol
• air dry completely

Probe:

for 10 slides prepare

• 180 µl formamide
• 120 µl 30% dextransulfate (Sigma D8906)
• 40 µl 20xSSC
• 10 µl salmon testes DNA (Sigma D9156)

21 µl of this + 4 µl of biotinylated probe

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

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

incubate over-night at 37°C 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 42°C, 20 min (temperature inside the jar!)

1 wash in 2xSSC at 42°C, 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 µl of a 1:100 dilution of Streptavidine-FITC (invitrogen #43-4311) (10 µl in 1 ml TBST 5% milk) on each slide

cover with parafilm

incubate in a humid chamber at 37°C for 1 hour

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

embed in 10 µl vectashield with 5µg/ml propidiumiodide

keep at 4°C until observation

• H. Hirai and P.T. LoVerde Parasitology Today, vol. 11, no. 8. 1995