90i microscope

Imaging was done as previously described^{12 (link)}. Briefly, third instar larvae were immobilized by cold treatment in glycerol, imaged with a NIKON 90i microscope, and the fluorescence intensity was quantified with ImageJ software.

VE DIC microscopy was done with 60×/1.00 NA water-immersion objective mounted on Nikon 90i microscope according to previously described (25 (link)). In 2-dpf live embryos, the neutrophil granule status is easy to be detected with DIC assay since the neutrophil granules are abundant and neutrophils are accessible to be observed by the lens.

For imaging sporozoite invasion and confocal imaging of developing exoerythrocytic forms, cells were visualized using a Nikon 90i microscope at 100× magnification, and images were acquired with a Hamamatsu Orca-ER camera using the Volocity 3D Image Analysis Software with image stacks deconvolved prior to combining and focused along the plane of the parasites. Epifluorescence imaging of exoerythrocytic forms was performed using an Olympus BX-53 upright microscope at 100× magnification. Imaging of hepatocytes following protein staining was performed using either a Zeiss Axioskop 2 microscope with a ProgRes MFcool camera using the ProgRes CapturePro software version 2.10.0.1 or using a BZ-X710 (Keyence, Osaka, Japan) All-in-One Fluorescence Microscope. BZ-X700 Analyzer Software (version 1.31.1) was used for Z-stack analyses to yield a single compressed fully focused image at 600× magnification from 9 to 11 planes, with each plane at 0.2 μm thickness.

Transgenic P. berghei ANKA parasites expressing P. falciparum MSP1 in place of the endogenous allele (de Koning-Ward et al., 2003 (link)) were grown in C57Bl/6 mice (Jackson Laboratories) housed under specific pathogen–free conditions. All animal work was performed with the approval of the University of Washington Institutional Care and Use Committee in accordance with the guidelines of the National Institutes of Health Office of Laboratory Animal Welfare. When parasitemia reached ∼5%, blood was harvested from euthanized animals by cardiac puncture, and red blood cells were washed and fixed in 4% paraformaldehyde with 0.0075% glutaraldehyde. Fixed cells were permeabilized with 10% Triton X-100 in PBS, blocked with BSA and incubated with MaliM03 IgM or IgG antibody along with a rabbit polyclonal anti-BIP antibody (kindly provided by Ashley Vaughan, Seattle Children’s Research Institute, Seattle, WA), followed by anti-rabbit antibody and anti-human IgM or IgG antibody conjugated to af488 and af594, respectively (Invitrogen). Finally, nuclei were stained with 6′-diamidino-2-phenylindole. PBS washes were performed between each step. Stained samples were applied to a slide and coverslipped using VectaShield. Images were obtained on a Nikon 90i microscope at 100× magnification.