Immunofluorescence assays

CHAPTER 2 General materials and methods

2.36 Immunofluorescence assays

Slides were coated with P. yoelii yoelii parasitized mouse red blood cells according to the method described by Perlmann et al. (1984).

The native P. yoelii yoelii protein PY05757 and P. yoelii yoelii LDH were probed with the anti-peptide antibodies on P. yoelii yoelii infected red blood cells. The protocol used was based on a method employed by Rasoloson et al. (2004). Anti-chicken antibodies conjugated with fluorescein isothiocyanate (FITC) were used to detect bound anti-peptide antibodies. The slides were counterstained with 4‟, 6-diamide-2‟-phenylindole dihydro chloride (DAPI) to indicate the parasite nuclear material.

2.36.1. Reagents

Coating buffer. Na₂CO₃ (0.0795 g) and NaHCO₃ (0.1465 g) were dissolved in approximately 40 ml dH2O and titrated to pH 9.6 with dilute HCl. NaN3 (0.01 g) was added and the volume made up to 50 ml.

Isotonic Tris buffer [0.15 M Tris, pH 7.2]. Tris (0.908 g) was dissolved in sterile dH2O (approximately 40 ml) and adjusted to pH 7.2 with HCl. The solution was then made up to 50 ml with sterile dH2O. As the solution was usually filter sterilized with a 0.22 μm acetate filter, the pH was adjusted to approximately pH 7.0 due to the tendency of solutions to increase in pH during the filtering process.

0.9% (w/v) NaCl. NaCl (0.9 g) dissolved in sterile dH2O (100 ml).

Hanks balanced salt solution (HBSS) [Sigma 9.5 g/l]. HBSS (0.95g) was dissolved in sterile dH2O (approximately 90 ml) by stirring gently on a magnetic stirrer. Sodium bicarbonate was added to a final concentration of 0.35 g/l. The solution was stirred until the sodium bicarbonate dissolved. The solution was then adjusted to pH 6.9 - 7.0 and made up to 100 ml with sterile dH₂O.

Tris buffered Hanks balanced salt solution (TBH). Isotonic Tris buffer, pH 7.2 (10 ml), 0.9%

(w/v) NaCl (90 ml) and Hanks balanced salt solution (HBSS) (100 ml) were combined and filter sterilize using a 0.22 μm acetate filter.

1% (v/v) Glutaraldehyde. 25% (v/v) Glutaraldehyde (200 μl) was made up to 5 ml with PBS.

0.05% (v/v) Tween-TBH. Tween-20 (50 μl) was diluted to 100 ml with TBH.

Phosphate buffered saline (PBS), pH 7.2. NaCl (8 g), KCl (0.2 g), Na2HPO4.2H2O (1.15 g) and KH2PO4 (0.2 g) were dissolved in dH2O (approximately 950 ml) and titrated to pH 7.2 if necessary. The volume was made up to 1 l with dH2O.

2% (w/v) BSA- PBS. BSA (0.5 g) was dissolved in PBS (25 ml).

2.36.2. Procedure

BALB/c mice were infected with P. yoelii yoelii parasitised mouse red blood cells by I.P. injection (Section 2.22). Blood was collected from the infected mice by retro orbital bleeding (Section 2.25) when the percentage of parasitized red blood cells in the mice was determined to be approximately 30% (Section 2.24). The blood was collected into Heparin coated Vacutainer^TM tubes and kept on ice. The volume of whole blood was determined and then the red blood cells were centrifuged (1 000 x g, 1 min). The plasma volume was determined in order to calculate the red blood cell packed volume. The red blood cells were suspended in a volume of TBH approximately equal to the whole blood volume and centrifuged as before to pellet the red blood cells. The supernatant was discarded and the packed red blood cells were suspended to 1% (v/v) haematocrit in TBH. The wells of 8 mm diameter, 8-welled slides were pre-treated with one drop of coating buffer per well (approximately 20 μl per well) for 30 minutes at room temperature. The coating buffer was then aspirated. One drop (approximately 20 μl) of the 1% (v/v) haematocrit was placed in each of the pre-treated wells and left for 30 minutes in order to allow the red blood cells to settle and adhere to the surface of the slide. Unbound red blood cells were washed off the slides by immersing them upside down in TBH and gently shaking them. The red blood cells were then fixed to the wet slides by covering them in a solution of 1% (v/v) glutaraldehyde in PBS. After 10 seconds the glutaraldehyde solution was allowed to run off the slides and the fixation procedure repeated. The slides were washed with sterile dH2O and allowed to air dry. The slides were stored at –20˚C until needed.

The wells of the slide coated with parasitized mouse red blood cells were blocked with 2% (w/v) BSA-PBS (20 μl per well, 1 h, RT). The blocking solution was aspirated and the wells washed with PBS (20 μl/well, 2 x 3 min). The PBS was aspirated and the wells were incubated with primary antibody (pre-immune IgY, anti-PfLDH IgY (controls) and anti- peptide FKLGSCYLYIINRNLKEI, CFKLGSCYLYIINRNLKEI and SDDDNRQIQDFEC IgY, 10 μg/ml in 2% (w/v) BSA-PBS, 1 h, RT). The primary antibody was aspirated and the wells were washed with PBS (20 μl/well, 3 x 5 min). The remainder of the protocol was carried out in the dark. Each well was incubated with secondary antibody (donkey anti- chicken-FITC conjugate diluted 1:100 in 2% (w/v) BSA-PBS, 20 μl/well, 1 h, RT). The wells

were washed with PBS (20 μl/well, 2 x 5 min) and stained with DAPI (5 μg/ml in PBS) (15 min, RT). The excess stain was rinsed off with PBS and the slides were stored overnight in the dark in a vacuum desiccator. Slides were viewed at a magnification of 1 000x using an Olympus AX70 fluorescent microscope. A green filter was used for FITC and a UV filter was used for DAPI. Digital photographs of the slides were taken with the CC12 Soft Imaging System™ colour camera and viewed with the analySIS™ software. Z-stack images were performed using the Confocal Zeiss LSM 710 microscope and ZEN light imaging software.

CHAPTER 3