Immunofluorescence Microscopy (Pf, fixed)

Curators

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Abstract

Use antibodies or sera to localize proteins to the surface of fixed, permeabilized 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.
• Fixative Solution
• Permeabilization Solution
• 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.
• 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!

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.

Discussion

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