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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.