IF Cytospun Slides (PJ Keller)

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IF Cytospun Slides

A.  Cytospin

-Spin cells at 20,000 to 50,000 cells per spot in 100-150 ml of media (preferably + at least 10% serum)

-Load 150-250 ml of sample into the chamber, spin at 500 rpm for 1’  with medium acceleration

-Fix spots in 100% ice cold methanol at –20 for 10’

-Air dry spots and freeze at –80 until use (better if used within 1-2 months, though I have stained years old slides as well with success)

B.  Immunofluorescence

  1. Allow slides to thaw at RT, dry any condensation with a kimwipe (avoiding spots, of course).  Circle spots with a pap pen.
  2. Permeabilize cells with 0.1% Triton-X 100 in PBS
    1. Add 150 ml per spot
    2. Incubate at RT 10’
    3. Wash 3X5’ in a coplin jar filled with PBS
  3. Block cells 1 hour in PBS + 1% BSA + 2% goat serum (for Alexa-fluor secondary antibodies)
    1. Add 150 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 ml per spot 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 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 ml per spot
    3. Incubate 5’ at RT in the dark, rinse with PBS 1X
  8. Mount slides with SlowFade kit and coverslip
    1. Blot excess PBS, add 1 drop equiibration buffer per spot
    2. Incubate at RT 5’, blot excess from the edge of the slide
    3. Add 1 drop slow fade solution per spot/well
    4. Cover with coverslips and seal with nail polish, store at 4C or –20 for longer term
    5. Clean backs of slides with windex before viewing