Basic Fluorescent in situ Hybridization (FISH)
实验概要
Fluorescence in situ hybridization method is a kind of physical map drawing method, use fluorescent element mark probe, to detect probe and split the chromosome or split between the middle period of the chromatin hybrid.
主要试剂
20x Saline-sodium citrate buffer [Details：SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7, or Product No. S6639].
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.
Ethanol.
Labeled probe. Plasmid DNA is labeled with biotin-11-dUTP using nick translation random priming or the polymerase chain reaction
Hybridization mix solution: 50% formamide [Details：Product No. F7508], 10% dextran sulfate [Details：Product No. D8906], 0.1% SDS [Details：Product No. L4390], 0.5-1.5 ng/µl labeled probe and 300 ng/ml Salmon Sperm DNA [Details：Product No. D7656] in 2x SSC.
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.
Antibody or detection compound [Details：e.g., Streptavidin-Cy3, Product No. S6402] in blocking buffer.
DAPI [Details：Product No. D9542] 2 µg/ml in antifade mounting medium.
主要设备
Heat block/ modified thermocycler.
Coplin jars for washing steps [Details：Product No. S6016, S5641 or S5891].
Fluorescence microscope, filters and optional triple band pass filter [Details：x58, Omega Optics].
实验材料
Plastic cover slips for incubation and hybridization steps [Details：cut from autoclavable waste bags, e.g., Product No. B4408]
Glass slides [Details：Product No. S8400].
实验步骤
1. Slide Preparation
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.
4) Rinse slides in 10 mM HCl.
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.
11) Air dry.
2. Hybridization
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.
3. Detection
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.
8) Incubate with 50 µl antibody or detection compound for 30-60 minutes (e.g., 5 µg/ml Streptavidin-Cy3 in blocking buffer).
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.
12) Analyze with fluorescence microscope.
