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Duolink® In Situ Short Instructions - Fluorescence

 

1. Blocking
Add blocking solution to samples.

Incubate.

• Remove block
2. Primary Antibodies
Dilute the primary antibodies in appropriate buffer and apply to samples.

Incubate.

• Wash in suitable buffer for 2 × 5 min
3. PLA® Probes
Dilute the two PLA probes 1:5 in appropriate buffer and apply to samples.

Incubate for 60 min at +37°C.

• Wash in 1x Wash Buffer A for 2 × 5 min
4. Ligation
Dilute the Ligation stock 1:5 in H2O. Dilute the Ligase at 1:40 in the solution and apply the mix to samples.

Incubate for 30 min at +37°C.

• Wash in 1x Wash Buffer A for 2 × 2 min
5. Amplification
Dilute the Amplification stock 1:5 in H2O. Dilute the Polymerase at 1:80 in the solution and apply the mix to samples.

Incubate for 100 min at +37°C.

• Wash in 1x Wash Buffer B for 2 × 10 min
•Wash in 0.01x Wash Buffer B for 1 min
6. Preparation for Imaging
Mount the samples with Duolink In Situ Mounting Medium with DAPI, wait for 15 min, and analyze in a fluorescence or confocal microscope.

Note: Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber.
Use volumes corresponding to your reaction area, see the Reaction Volume Guide at sigma.com/duolink.
Use a freezing block when removing the enzymes from the freezer (-20ºC).
Washing should be done in a minimum volume of 70 ml on a shaker with gentle orbital shaking.

 

Materials

     
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