Fix and permeabilize the samples as follows:

Blocking and antibody incubation

Incubate the samples with a blocking solution for 1 hour at 37°C in a humidified chamber.
Prepare the primary antibodies in antibody diluent and incubate the samples overnight at 4°C.

Wash the samples twice with Buffer A for 5 minutes each.
Incubate the samples with PLA probes (anti-mouse MINUS and anti-rabbit PLUS) prepared in antibody diluent for 1 hour at 37°C.

Wash the samples twice with Buffer A for 5 minutes each.
Perform the ligation step by incubating the samples with ligase enzyme diluted in ligation buffer for 30 minutes at 37°C in a humidified chamber.
Wash the samples twice with Buffer A for 5 minutes each.
Incubate the samples with polymerase enzyme diluted in amplification buffer for 100 minutes at 37°C in a humidified chamber.
Wash the samples twice with Buffer B for 10 minutes each.
Perform a final wash with 0.01% Buffer B for 1 minute.

Mount the slides with Duolink in situ mounting medium containing DAPI.
Imaging and Storage
Wait 15 minutes before imaging using a fluorescence or confocal microscope with at least a 20x objective.
After imaging, store the slides:
In the dark at 4°C for up to 4 days.
Or at -20°C for up to 6 months.

Perform negative control experiments by incubating the samples with only one or none of the primary antibodies before adding the PLA probes.

The kit is stored all over RT, 4C and -20C

4F4 fridge in freezer room with glass room, box in the middle shelf filled with smaller boxes, “Duolink” original boxes contain: