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
Loading
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.
Sign In To Continue
To continue reading please sign in or create an account.
Don't Have An Account?