8 In Situ Hybridization for RNA: Radioactive RNA Probe
123
4. Immerse the holder in one of four staining dishes (prewarmed at 50° C) and shake for 20 mins. Repeat this process
three times.
5. Prewarm ISHR9 at 37° C without RNase A.
6. Prewarm final washing solution of ISHR11.
7. Wash the slides in ISHR9 for RNase treatment.
S. Incubate the slides in ISHR9 after addition of ISHRlO containing RNase A at 37° C for 30 mins exactly.
9. Wash the slides with ISHR9 used in the previous step 7 for
5 mins.
10. Wash the slides in ISHRll containinglO mM DTT for 20
mins at 50° C three times.
11. Dehydrate the samples by dipping sequentially for 3 mins
each in: 70% ethanol, 90% ethanol,100% ethanol, xylene,
100% ethanol, and 100% ethanol.
12. Air dry the slides.
1. Prepare silica-gel, a light-tight box, black vinyl bags, a sheet
of black curtain for wrapping the box, black tape.
Note: All of the following and developing steps must be carried
out in a dark room using a safe-light.
2. Prepare stock emulsion (Kodak NTB-2) by dispense into 109
aliquots and store them in plastic light -tight containers (e.g.,
black film container) wrapped in aluminium foil, and store
at 4° C.
3. Melt stock emulsion in water bath at 42° C and gently pour
10ml prewarmed water (1:1 dilution) in the container. Mix
gently by swirling to avoid making bubbles.
4. Pour emulsion in the dipping chamber immersed in the
water bath at 42°C.
5. Dip two test slides to remove the surface bubbles.
6. Immerse a pair of the slides (back to back) in emulsion at
42° C and withdraw them slowly.
Autoradiography
123
4. Immerse the holder in one of four staining dishes (prewarmed at 50° C) and shake for 20 mins. Repeat this process
three times.
5. Prewarm ISHR9 at 37° C without RNase A.
6. Prewarm final washing solution of ISHR11.
7. Wash the slides in ISHR9 for RNase treatment.
S. Incubate the slides in ISHR9 after addition of ISHRlO containing RNase A at 37° C for 30 mins exactly.
9. Wash the slides with ISHR9 used in the previous step 7 for
5 mins.
10. Wash the slides in ISHRll containinglO mM DTT for 20
mins at 50° C three times.
11. Dehydrate the samples by dipping sequentially for 3 mins
each in: 70% ethanol, 90% ethanol,100% ethanol, xylene,
100% ethanol, and 100% ethanol.
12. Air dry the slides.
1. Prepare silica-gel, a light-tight box, black vinyl bags, a sheet
of black curtain for wrapping the box, black tape.
Note: All of the following and developing steps must be carried
out in a dark room using a safe-light.
2. Prepare stock emulsion (Kodak NTB-2) by dispense into 109
aliquots and store them in plastic light -tight containers (e.g.,
black film container) wrapped in aluminium foil, and store
at 4° C.
3. Melt stock emulsion in water bath at 42° C and gently pour
10ml prewarmed water (1:1 dilution) in the container. Mix
gently by swirling to avoid making bubbles.
4. Pour emulsion in the dipping chamber immersed in the
water bath at 42°C.
5. Dip two test slides to remove the surface bubbles.
6. Immerse a pair of the slides (back to back) in emulsion at
42° C and withdraw them slowly.
Autoradiography