Counterstaining after the Duolink In Situ Protocol

We recommend applying the counterstaining protocol after the completion of the Amplification step in section 7.3, step 5 of the Duolink In Situ Fluorescence User Manual. The following protocol exemplifies counterstaining using a directly labeled FITC anti-alpha tubulin antibody:

Perform all the steps at room temperature.

1. Tap off the Amplification-Polymerase solution from the slides.
2. Wash the slides in 1x Wash Buffer B for 2 x 10 min.
3. Move the slides to 1x Wash Buffer A for 1 min
4. Proceed to the counterstaining protocol and incubate the sample with e.g. a FITC labeled mouse anti-alpha tubulin for 40 min. - If the antibody used for counterstaining requires an antibody diluent different from that used during the Duolink assay, it is advised to add a blocking step before incubation with the counterstaining antibody
5. Wash the slides with 1x Wash Buffer A for 2 x 2 min.
6. Wash the slides with 0.01x Wash Buffer B for 1 min.
7. Mount the slides following the protocol in section 7.3, step 7 in the Duolink In Situ Fluorescence User Manual.

Above: Single recognition of HER2 in SK-BR-3 cells. Red: PLA signals, each representing one HER2 protein. Green: counterstain of alpha tubulin. Blue: nucleus. PLA probes anti-Rabbit PLUS and MINUS, Duolink In Situ Detection Reagents Orange.

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