IF on Chamber Slides (PJ Keller)

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IF on Chamber Slides

A.  Culture

-Plate 5000-10,000 cells per well (8 or 4-well chamber slides, respectively) in growth media

-Allow cells to grow until 50-70% confluent

B.  Immunofluorescence (note: smaller volumes are recommended for 8-well chamber slides, larger for 4-well; regardless of the exact amount put on the cells, make sure that there is enough liquid to cover the cells completely as drying out can lead to high background staining)

  1. Fix cells in Methanol
    1. Aspirate media from cells
    2. Wash wells with PBS 1X
    3. Add 250-500 µl cold methanol
    4. Incubate at –20 C for 10 min
    5. Wash wells with PBS 1X
  2. Permeabilize cells with 0.1% Triton-X 100 in PBS
    1. Add 150 to 250 ml per well
    2. Incubate at RT 10’
    3. Wash 3X5’ with PBS
  3. Block cells 1 hour in PBS + 1% BSA + 2% goat serum (for Alexa-fluor secondary antibodies)
    1. Add 150 to 250 ml per spot
    2. Incubate at RT 1 hour in a humidified chamber
  4. Add primary antibodies overnight at 4C or 1 hour at RT
    1. For Rabbit aCK14 use at 1:500 in PBS + 1% BSA
    2. For Mouse aCK18 use at 1:500 in PBS + 1% BSA
    3. Prepare a solution of both antibodies at 1:500 to double stain
    4. Add 150 to 250 ml per well and incubate in a humidified chamber
  5. Wash slides 3×5’ in PBS to remove residual primary antibody
  6. Add secondary antibodies for 1 hour at RT
    1. Use goat anti-Rabbit or goat anti-Mouse Alexa fluor conjugated antibodies (Alexa 488 is green, Alexa 555 or 546 are red-orange)
    2. Prepare secondaries at 1:500 in PBS + 1% BSA (prepare a solution of both mouse and rabbit secondary antibodies to double stain)
    3. Add 150 to 250 ml per spot and incubate in a humidified chamber (in the dark!)
    4. Wash 3×5’ with PBS in the dark
  7. DAPI counter stain the nuclei
    1. Dilute 25 mg/ml stock in 1:100 PBS (i.e. 1 ml PBS + 10 ml DAPI)
    2. Add 150 to 250 ml per spot
    3. Incubate 5’ at RT in the dark, rinse with PBS 1X
  8. Mount slides with SlowFade kit and coverslip
    1. Remove the chambers with the provided tools
    2. Blot excess PBS, add 1 drop equilibration buffer per well
    3. Incubate at RT 5’, blot excess from the edge of the slide
    4. Add 1 drop slow fade solution per well
    5. Cover with coverslips and seal with nail polish, store at 4C or –20 for longer term