From 2013.igem.org

IMMUNOFLUORESCENCE

Aspirate the medium from wells. Wash wells with PBS 1000 µl/well. Fix cells with prewarmed 4% Paraformaldehyde/PBS 500 µl /well for 15 min.at RT (add very slowly). Remove supernatant. Treat the cells for 15 min. at RT with prewarmed (37oC) TZN buffer 500 µl/well (add very slowly). Slowly mix on shaker. Aspirate supernatant. Wash wells with PBS, 750 µl/well, 5 min X 3 on shaker. Add Blocking Solution 500 µl/well, incubate for 1 hr at RT. Mix slowly on shaker.

Blocking Solution (f) NGS 10% BSA (10%) 1% PBS-Tx 0.3%

Aspirate off the blocking solution. Add 100 µl/ well 1st Ab. mixture. Seal the plate with parafilm, incubate at RT for 2 hr or o/n @ 4oC. Wash with PBS-TX 0.3%, 750 µl/well, 5 min X 1 on rocking shaker. Wash with PBS 750 µl/well, 5 min X 2 on rocking shaker. Add 100µl/ well 2nd Ab. Work in dark from this point! Incubate the plate at RT for 1 hr. Wash with PBS 1ml/ well , 5 min X 3 on rocking shaker. Add 300 µl/well TO-PRO-3 (1 µM) (light sensitive!)

Incubate at RT for 15 min. Wash with PBS 1 ml/well, 5 min X 3 on rocking shaker. Add 1 drop of Prolong Gold Mounting Medium onto slides for each coverslip. Take out coverslip from the well. Invert and put on mounting soln. on the slide. Seal coverslip with nail polish. Let the coverslip dry. Slides can be stored at 4oC for a long time (at dark). Take images with confocal microscope.