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HomeProtein ExpressionDuolink® In Situ Short Instructions - Fluorescence

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.
Materials
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Notes:

  • Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber.
  • Use a freezing block when removing the enzymes from the freezer (-20 ºC).
  • Washing should be done in a minimum volume of 70 mL on a shaker with gentle orbital shaking.
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