Place 0.5ml of cells into each well of 2 LabTek® 4-chamber slides (Fisher
Cat.#12-565-21). Allow to grow overnight as usual.
The next morning , remove medium from wells, fix cells on slides with 0.5ml/well 4%
paraformaldehyde in PBS at ambient room temperature for 60min.
Wash cells fixed on slides in 1XPBS for 3 times for 5 minutes each at ambient room
temperature.
Permeablize the cells with 0.1% Triton® X-100 in PBS for 10 minutes.
Wash slides in 1XPBS 3 times for 5 minutes each at ambient room temperature.
Block the cells with 1% normal donkey serum [Jackson ImmunoResearch Cat.#017-000-121] in
PBS for 45 minutes at ambient room temperature.
Incubate with 0.5ml/well Anti-Luciferase pAb (G7451) at 20µg/ml or 0.5ml/well
non-immune goat IgG [Jackson ImmunoResearch Cat.#005-000-003] at 20µg/ml in PBS with 1%
donkey serum for 2 hour at ambient room temperature. For secondary control, add 0.5ml PBS
with 1% donkey serum only. Concentration of antibody may need to be optimized for
different cell lines and luciferase expression levels.
Wash slides in 1XPBS 3 times for 15 minutes each at ambient room temperature.
Incubate with 1:200 dilution of anti-goat IgG-FITC (Jackson ImmunoLabs, Cat.
705-095-147) in PBS with 1% donkey serum for 60 minutes at ambient room temperature. Keep
slides in dark or wrap with foil.
Wash slides in 1XPBS 3 times for 15 minutes each at ambient room temperature and keep
the slides in dark or wrap with foil while washing.
Drain the liquid and mount the slides in Vectashield with DAPI (Vector Labs, Cat.#
H-1200) by adding 30-50µl to the slide and carefully placing a 22mm x 50mm glass
coverslip over the wells, avoiding bubbles.
