Immunoflourescence of Cultured Cells

  • 来源:生物谷
  • 时间: 2010/5/3
  • 浏览人数: 4765

    Materials
    L-lysine coated glass cover slips or charged glass slides
    Neutral Buffered Formalin (Sigma HT50-128)
    0.5% NP40 in PBS
    (2.5 N HCl or 0.07 N NaOH for BrDU staining only)
    Primary and secondary antibodies
    Vectashield (Vector Labs)

    Protocol

    1) Grow cells on L-lysine coated glass slips or cytospin cells onto charged glass slides.

    2) Fix cells for 5 min. in neutral buffered formalin.

    3) Permeabilize the nucleus by incubating in 0.5% NP40 in PBS at r.t.

    4) Rinse in 3 changes of PBS for a total of 10 minutes.

    5) For BrDU staining denature the DNA by one of the following:

    a) soak in 2.5N HCl at 37°C for 15 min, or

    b) 0.07N NaOH for 2 min at room temp

    5) Add 100 µL primary antibody (titer determined emperically ~10x the concentration used in a western). Cover with a glass slip and place in a humidified chamber at r.t. for 1 hr.

    6) Float the coverslip off by dipping into a jar of PBS, and rinse as in 4).

    7) Add secondary antibody as in 5) and 6) above. (e.g. FITC-conj goat anti-rabbit (1:1000) or Biotinylated isotype specific anti mouse for immunoperoxidase staining). Wash as before.

    8) To minimize quenching of flourochrome mount with Vectashield (Vector labs) and cover with a glass slip.