Immunocytochemistry with monolayer cell culture slides

Dominik Haudenschild and David M. Johnson

September 27, 1993 at CBRC

 

•Very carefully remove medium from slide. Use vacuum and pasteur pipette attachment. Cells may detach depending upon cell type, length of growth period, and type of slide. Avoid plastic.

•3 washes in PBS. 5 min. each. Place PBS onto wells carefully using pasteur pipetteUse enough to sufficiently cover cells. Vacuum carefully after every wash. This is the same for all following washes.

Optional fixation methods:

Acidic alcohol:

•Freeze slides at -20oC with 70% ethanol, 50 mM glycine pH 2.0 for a minimum of 20 minutes. Vacuum carefully when done.

•3 washes in PBS. 5 min. each.

 

PFA (Paraformaldehyde):

•4% PFA in PBS 20 min. Prepare 4% PFA in sufficient amount. Use PBS, add appropriate amount of PFA (mixture will be cloudy since PFA doens't dissolve well in PBS). Heat mixture carefully to near boiling and solution will become clear. 10mL is usually adequate for all slides. Prepare this fresh every time. Vacuum carefully when done.

•3 washes in PBS. 5 min. each.

•0.1 M glycine 30 min. Wash cells in glycine. Use enough to ensure that cells are covered.

•3 washes in PBS. 5 min. each.

 

•Incubate with 1o antibody at37oC for 30 min.. Place on 30-50mL of the appropriate antibody. Do the same for multiple antibodies, but make sure that the antibodies are from different sources (ie. one rabbit and one mouse). Skill is required, when using the smaller volumes, in spreading out the antibody to cover all of the cells and not damage them. Patience and a steady hand is needed. A 1:200 anitbody dilution is usually sufficient, although concentration is antibody and researcher dependent. Incubation can also be done at r.t. for 1 1/2 hours or O/N at 4oC, however be carefull not to let cells dry out (soaking incubation chamber with PBS is a good way to avoid this).

•3 washes in PBS. 5 min. each.

 

•Incubate with 2o antibody at 37oC for 30min. Prepare secondary antibody use donkey a-mouse rhodamine and a-rabbit flourescein (if using mouse and rabbit primaries). Prepare in 1%BSA in PBS. Add in Hoechst nuclear dye at a 1:1000 dilution ( 1µL per 1 mL). Spin antibodies at 14,000 rpm for 3-4 minutes to precipitate out particles that may give a high background. Put antibodies on slides in 30-50mL drops. Donkey antibodies must be used because they don't cross react -- unlike bought a-mouse or a-rabbit antibodies.

•3 washes in PBS. 5 min. each.

 

•Mount. Mount coverslips in 95:5 glycerol: PBS + 0.5mg/mL Na azide.