Basic Fluorescent in situ Hybridization (FISH)
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实验试剂
RNase A [ Details ： Product No. R4642] 100 µg/ml in 2x SSC.
Pepsin [ Details ： Product No. P6887] 40 units/ml in 10 mM HCl.
Paraformaldehyde, EM grade [ Details ： Product No. P6148] freshly depolymerized, 4% w/v in water.
Wash buffer: 20% formamide [ Details ： Product No. F7508] in 0.1x SSC.
Detection buffer: 0.2% Tween 20 [ Details ： Product No. P1379] in 4x SSC.
Blocking buffer: 5% bovine albumin [ Details ： Product No. A3803] in detection buffer.
DAPI [ Details ： Product No. D9542] 2 µg/ml in antifade mounting medium.
实验设备
Heat block/ modified thermocycler.
Coplin jars for washing steps [ Details ： Product No. S6016, S5641 or S5891].
实验材料
Glass slides [ Details ： Product No. S8400].
实验步骤
1) Start with chromosome preparations from any cell type.
2) Incubate with 200 µl RNase for 1 hour at 37 °C
3) Wash slides in 2x SSC for 5 minutes, repeat.
5) Incubate with 200 µl pepsin for 10 minutes at 37 °C.
6) Rinse slides in deionized H2O.
7) Wash slides in 2x SSC for 5 minutes, repeat.
8) Stabilize slides in paraformaldehyde for 10 minutes.
9) Wash slides in 2x SSC for 5 minutes, repeat.
10) Dehydrate slides in an ethanol series: 70%, 80%, 95%; 2 minutes each.
Prepare 30 µl hybridization solution per slide. Heat to 70 °C. for 10 minutes and place on ice.
Place 30 µl of hybridization solution on each slide and cover with a plastic cover slip.
Denature slide at 65-70 °C for 5 minutes on heat block.
Gradually decrease temperature to 37 °C.
Hybridize at 37 °C overnight in humidity chamber.
1) Wash slides in 2x SSC to remove coverslip.
2) Wash slides in wash buffer at 40 °C for 5 minutes, repeat.
3) Wash slides in 0.1x SSC at 40 °C for 5-15 minutes.
4) Wash slides in 2x SSC at 40 °C for 5-15 minutes.
5) Cool slides to room temperature.
6) Equilibrate slides in detection buffer for 5 minutes.
7) Block in blocking buffer for 20-30 minutes.
9) Wash slides in 2x SSC for 5 minutes, repeat twice.
10) Counterstain with DAPI solution for 10 minutes.
11) Rinse briefly and mount in antifade mounting medium.
