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.