Immunofluorescence Microscopy (Pf, live)

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Abstract

Use antibodies or sera to localize proteins to the surface of live, intact Plasmodium falciparum infected red blood cells.


Reagents

  • 1% gelatin (w/v) in RPMI
  • RPMI Media
    • Store at 4C, warm up to 37 C right before use.
  • PBS
    • Phosphate buffered saline, keep on ice.
  • Blocking Buffer
    • 3% Bovine Serum Albumin (BSA) (w/v) in PBS
    • 1.5 g in 50 mL, make fresh, sterile filter, and keep on ice during use.
  • Antibodies
  • DAPI, hoechst dye, or some other membrane permeable DNA dye (to stain nucleus)
  • Glass Coverslip
    • No. 1.5 (0.17 mm, 0.16 - 0.19 mm) thickness is best for DeltaVision Deconvolution microscope
  • Microscope Slide
  • Nail Polish
    • To seal coverslips on microscope slide
  • Mounting Medium
    • Right before using, place 2-3 crystals p-phenylenediamine (~1 mm diameter) in a microcentrifuge tube and add 100 uL water. Vortex and let sit in dark for 5 minutes. Centrifuge briefly and use the supernatant as antifade solution. Make fresh every time.

Equipment

  • Heat block set to 37 C
  • Refrigerated microcentrifuge, or a microcentrifuge placed in a cold room (4C)
  • Bunsen burner

Notes

  1. In this protocol, examples assume 12 mL (1 plate) of culture that is gelatin purified, which will be suspended in a 1 mL volume after purification.
  2. All centrifuge steps are at 2000 rpm in the microcentrifuge for 2 minutes at 4C.
  3. NEVER vortex to resuspend cells. If need be, gently flick the tube or pipet slowly up and down with a 1 mL pipet tip.
  4. Be gentle with cells! Keep them on ice at all times!
  5. Keep all solutions with fluorescent dye in the dark whenever possible!

Procedure

  1. Gelatin purify 12 mL of parasite culture to enrich for troph and schizont stage parasites
    1. Thaw an aliquot of frozen 1% gelatin at 37C. Use two tubes for 12 mL of culture.
    2. Spin culture at 1400 rpm (394 g) for 5 minutes with a brake of 1 (low) and discard supernatant.
    3. Add a volume of RPMI that is equal to the volume of the pellet. (12 mL culture at 4% hematocrit should be 480 uL pellet)
    4. Add four times the org. pellet volume of 1% gelatin and aliquot 1 mL volumes to 1.5 mL eppendorf tubes.
      1. For example, if pellet is 480 uL, add 480 uL RPMI and mix. Aliquot 240 uL of this mixture into each of four tubes. Add 960 uL of 1% gelatin to each of the tubes.
    5. Incubate at 37C for 10 minutes (Use different times for different applications, e.g. 20 minutes to get really pure trophs). Uninfected blood and ring stage infected red blood cells (iRBCs) will sediment in the pellet and mature stage iRBCs remain in the suspension.
    6. Transfer upper layer to another tube.
    7. Wash the pellet and upper suspension three times with PBS.
      1. Washing can take place in multiple tubes, but in the end, the 12 mL of culture after gelatin purification should yield roughly 40 uL of pellet. Resuspend this in a final volume of 1 mL to roughly recreate 4% hematocrit.
  2. Smear the iRBCs from the pellet and upper suspension to determine percentage of parasites.
  3. Block parasites with 2% BSA (w/v) in PBS for 45 minutes on ice or at 4C by resuspending 2 uL pellet in 200uL blocking buffer.
  4. Centrifuge parasites and remove blocking buffer.
  5. Aliquot 5 uL of parasite pellet per slide into separate tubes, then add 5 uL of primary antibody to make it a 1:2 dilution of antibody. You can also add 1 uL of antibody and 4 uL of 2% BSA (w/v) in PBS to make it a 1:10 dilution. Incubate at 4C for 1 hr with slow rotation.
  6. From now on, the instructions will be for each tube.
  7. Wash cells 3 times with 200 uL of 2% BSA (w/v) in PBS.
  8. Resuspend parasites in 25 uL of secondary antibody solution, which should consist of secondary antibody at 1:25 and 1:500 of 10 mg/mL DAPI, and incubate at 4C for 30 min with slow rotation. Don’t add the DAPI in this step if you will do a tertiary antibody incubation.
  9. Wash cells 3 times with 200 uL of 2% BSA (w/v) in PBS.
  10. (Optional) Resuspend parasites in 25 uL of tertiary antibody solution, which should consist of tertiary antibody at 1:25 and 1:500 of 10 mg/mL DAPI, and incubate at 4C for 30 min with slow rotation.
  11. Wash cells 3 times with 200 uL of 2% BSA (w/v) in PBS.
  12. Add cells: Pipet 1 uL of pellet resuspended in 5 uL 2% BSA (w/v) in PBS on the center of the coverslip and let sit for a few minutes.
  13. Add mounting media: Add 1 uL to the middle of the coverslip and mix by pipetting
    1. Don’t mount multiple slides at the same time. If you have multiple samples, do one and keep the other samples on ice.
  14. Add Slide: Flame a slide, let it cool, then place over the coverslip. Keep slides in dark as much as possible to prevent photobleaching of dyes.
  15. Seal slide by painting the edges of the coverslip with nailpolish or Valap (1:1:1 vaseline:lanolin:paraffin wax) and let the nailpolish dry in the dark.
  16. Go to the microscope and take some pretty pictures!

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