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Cell IMF Protocols

FIXATION STEPS 1-4 performed on ICE and ALL liquids should be at 4oC:

  1. Place coverslip(s) with cells in 6-well plastic petri dish(es) and place dishes on ice.
  2. Rinse coverslip(s) 1x with PBS.
  3. Add 1.0 mL ice cold 4% PFA/PBS, pH 7.2 to each coverslip for 1 min. Add 1.0 mL ice cold methanol to each cs and gently mix after each addition. Quickly add an additional 2.0 mL of methanol to each cs. Aspirate liquid and add another 2.0 mL methanol, aspirate again and quickly add another 2.0 mL. Let cells sit on ice for another 10 min.
  4. Aspirate methanol and quickly replace with PBS. Rinse cs with PBS 2-3x and rehydrate cells by incubating with 3 changes of PBS for 5 min each (3 x 5 min).

IMMUNOSTAINING (done at room temp with room temp. liquids)

  1. Block coverslip(s) with 1.0 mL of 1% BSA/PBS for 15 - 30 min.
  2. Place coverlip(s) in a humidified chamber. This is usually a large plastic petri dish with moistened filter paper in the bottom upon which parafilm is placed. As you place the cs into the chamber, remove excess liquid by placing the edge of the cs gently against a Kim-Wipe. This will wick the fluid off. Add ~100uL of 1o antibody(ies) diluted to the desired concentration in 1% BSA/PBS to each cs and incubate for 1h. Be sure not to let the cs dry out as you are adding the antibody solutions!
  3. Place cs back in 6-well dish and wash 3x5 min in 0.1% BSA/PBS.
  4. Place cs back in humidified chamber as above and incubate each coverslip with 100uL of of 2o antibody(ies) diluted in the desired concentration in 1% BSA/PBS for 30 min.
  5. Place cs back in 6-well dish and wash 2x5 min in 0.1% BSA/PBS and 1x5 min in PBS.
  6. Remove excess liquid and mount coverslip(s) on glass slides with mounting media and seal edges of coverslip(s) with nail polish. Allow cs to dry at least 15 min before viewing on microscope.

Options:

  1. Use 0.2% BSA/PBS instead of 0.1% BSA/PBS for rinses during immunostaining.
  2. Blocking may be done at 4oC.

Alternate IMF Protocol for Methanol sensitive antibodies

WIFB cells: Pre-extract with 0.2% Tx100 in PHEM buffer

Reagents:

All steps performed at room temperature

  1. Rinse cells 2X with PBS
  2. Permeabilize in 0.2% TX100/PHEM for EXACTLY 2min

(Handle cells GENTLY from this point on - they can easily float off the coverslip)
  1. Briefly rinse 2X in PBS
  2. Fix for 30min in 4% PFA/PBS
  3. Rinse 3X 5min in PBS
  4. Block for 30min in 1% BSA/PBS
  5. Incubate for 1hr in primary antibody (diluted in 1%BSA/PBS)
  6. Rinse 3X5min in 0.2% BSA/PBS
  7. Incubate for 30 min in secondary antibody (diluted in 1% BSA/PBS)
  8. Rinse 2X5min in 0.2% BSA/PBS
  9. Rinse 1X5min in PBS
  10. Mount coverslips on glass slide