Teflon printed 12 well slides

NF54 Pfspz, or cultures of iRBCs at 6% parasitaemia were washed twice in PBS/1% BSA. Teflon printed 12-well slides (Electron Microscopy Sciences, Hatfield, PA) were coated with Pfspz (5000/well) or with iRBC (10,000 RBCs/well at 6% parasitaemia) as described [10 (link)]. Slides were air dried and stored at − 80 °C until use. Upon thawing, slides were blocked with PBS/1% BSA for 30 min at 37 °C. Twenty microliters of plasma starting at 1:20 and sequential two-fold serial dilutions were added to the wells and incubated for 1 h at 37 °C. Slides were washed three times with PBS, incubated with FITC-labeled goat F(ab′)₂ anti-human IgG (γ chain) or anti-human IgM (µ chain) (Southern Biotech) to measure specific human IgG or IgM antibody titers, or with anti-human IgG (H + L) (BDbiosciences) to measure total specific human antibody titers, for 30 min at 37 °C, washed, and mounted with Vectashield-DAPI (Vector Laboratories).

Teflon printed 12-well slides (Electron Microscopy Sciences, Hatfield, PA) were coated with 3×10⁴P. marinus parasites in 1× PBS/1%BSA/well, air-dried and stored at −80°C until use. Slides were thawed for 30 min at room temperature and then blocked with 1xPBS/1% BSA for 30 min at 37°C. Serum samples at various dilutions (two-fold dilution series starting at 1∶20) were added to the parasite-coated wells and incubated for 1 h at 37°C. Slides were washed three times with 1xPBS, incubated with FITC-labeled F (ab’)₂ goat anti-mouse IgM, IgG, or IgA (Southern Biotechnologies, Birmingham, AL) at a 1∶40 dilution in 1xPBS/0.1% Evans blue for 30 minutes at 37°C. Slides were washed, air dried, and mounted with VECTASHIELD-DAPI (H-1200, Vector laboratories, Burlingame, CA).

Teflon printed 12-well slides (Electron Microscopy Sciences, Hatfield, PA) were coated either with P. falciparum (3D7) sporozoites (5,000 sporozoites per well/2% bovine serum albumin (BSA, Sigma) or parasite infected-red blood cells (iRBC) prepared 1:20 dilution from the final suspension and dispensed 10 µl per well. Slides were air dried and stored at −80°C until ready to use. Upon thawing, slides were blocked with PBS/1% BSA for 30 min at 37°C. Serum at two-fold dilutions was added to the wells and incubated for 1 h at 37°C. Slides were washed three times with 1xPBS and incubated with fluorescein-labeled anti-monkey IgG (Seracare Life Sciences Inc, Milford, MA) secondary antibody for 30 min at 37°C. Washed slides were mounted with Vectashield-DAPI (Vector Laboratories, Burlingame, CA) and examined under Optical UV microscope. IFA results were reported as end point dilution representing last serum dilution at which fluorescence was scored as positive.