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Basic Fluorescent in situ Hybridization (FISH)

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953

实验试剂

 

 

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.

 

实验设备

 

 

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.

 

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