The recombinant CIDRa2.1 domain of PF3D7_0617400 (MAL6P1.252) was produced at the Statens Seruminstitut, Cophenhagen, Denmark as described in [18 (link)] and used to immunize mice. Blood of immunized mice was sent to the Institute of Tropical Medicine, Tübingen, Germany. Mouse sera containing IgG α PF3D7_0617400 mouse secondary antibody were depleted from RBCs by filtering through a MN 615 ¼ filter. IgG was purified using Protein G Spin Columns from Thermo Scientific according to the manufacturer’s protocol.
For IFA, very thin blood smears of parasite cultures were fixated in − 20 °C cold 100% methanol for 5 min and then stored at − 20 °C until further use. IFA was performed following a modified protocol as previously described [29 (link)].
After a 5 min rehydration step in 1xPBS, slides were incubated for 1–2 h with anti CIDRa2.1 PF3D7_0617400 (diluted 1:50 in 1xPBS/1%BSA). The slides where then washed 3× with 1xPBS and incubated for 1 h with Alexa488 coupled mouse sera IgG α PF3D7_0617400 mouse secondary antibody (not diluted, 0.44 mg/ml). After another 3× washing with 1xPBS, slides were stained with Hoechst 33342 (diluted 1:1000) for 30 min. Slides were mounted over night with MOWIOL-488 and viewed through 100× oil immersion lens at a fluorescent microscope.
For IFA, very thin blood smears of parasite cultures were fixated in − 20 °C cold 100% methanol for 5 min and then stored at − 20 °C until further use. IFA was performed following a modified protocol as previously described [29 (link)].
After a 5 min rehydration step in 1xPBS, slides were incubated for 1–2 h with anti CIDRa2.1 PF3D7_0617400 (diluted 1:50 in 1xPBS/1%BSA). The slides where then washed 3× with 1xPBS and incubated for 1 h with Alexa488 coupled mouse sera IgG α PF3D7_0617400 mouse secondary antibody (not diluted, 0.44 mg/ml). After another 3× washing with 1xPBS, slides were stained with Hoechst 33342 (diluted 1:1000) for 30 min. Slides were mounted over night with MOWIOL-488 and viewed through 100× oil immersion lens at a fluorescent microscope.
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